Coupling endonucleases with end-processing enzymes drives high efficiency gene disruption

ABSTRACT

The present disclosure relates to the co-expression of an endonuclease with an end-processing enzyme for the purpose of enhanced processing of the polynucleotide ends generated by endonuclease cleavage.

RELATED APPLICATION

The present application is a divisional application of U.S. applicationSer. No. 14/173,705, filed on Feb. 5, 2014, which is a divisionalapplication of U.S. application Ser. No. 13/405,183, filed on Feb. 24,2012, now issued as U.S. Pat. No. 8,673,557, which in turn claimspriority to U.S. provisional application No. 61/447,672, filed Feb. 28,2011, pursuant 35 U.S.C. 119(e), and the disclosure for each of theserelated applications which is incorporated by reference herein in theirentirety.

STATEMENT REGARDING FEDERALLY SPONSORED R&D

This invention was made with government support under Grant No. T32GM07270 awarded by the U.S. National Institute of General MedicalSciences and Grant Nos. RL1CA133832, UL1DE019582, R01-HL075453,PL1-HL092557, RL1-HL092553, RL1-HL92554, and U19-AI96111 awarded by theNational Institutes of Health.

FIELD

The present disclosure relates to molecular and cellular biology. Someembodiments relate to genome engineering and the introduction oftargeted, site-specific DNA breaks mediated by endonucleases to achievegene disruption or site-specific recombination. Several embodimentsrelate to compositions and methods for partial or complete inactivationof a target gene. Some embodiments relate to inactivation of a targetedgene for therapeutic purposes and/or to produce cell lines in which atarget gene is inactivated.

BACKGROUND

Targeted gene disruption has wide applicability for research,therapeutic, agricultural, and industrial uses. One strategy forproducing targeted gene disruption is through the generation ofdouble-strand DNA breaks caused by site-specific endonucleases.Endonucleases are most often used for targeted gene disruption inorganisms that have traditionally been refractive to more conventionalgene targeting methods, such as algae, plants, and large animal models,including humans. For example, there are currently human clinical trialsunderway involving zinc finger nucleases for the treatment andprevention of HIV infection. Additionally, endonuclease engineering iscurrently being used in attempts to disrupt genes that produceundesirable phenotypes in crops.

The homing endonucleases, also known as meganucleases, are sequencespecific endonucleases that generate double strand breaks in genomic DNAwith a high degree of specificity due to their large (e.g., >14 bp)cleavage sites. While the specificity of the homing endonucleases fortheir target sites allows for precise targeting of the induced DNAbreaks, homing endonuclease cleavage sites are rare and the probabilityof finding a naturally occurring cleavage site in a targeted gene islow.

One class of artificial endonucleases is the zinc finger endonucleases.Zinc finger endonucleases combine a non-specific cleavage domain,typically that of FokI endonuclease, with zinc finger protein domainsthat are engineered to bind to specific DNA sequences. The modularstructure of the zinc finger endonucleases makes them a versatileplatform for delivering site-specific double-strand breaks to thegenome. One limitation of the zinc finger endonucleases is that lowspecificity for a target site or the presence of multiple target sitesin a genome can result in off-target cleavage events. As Fok1endonuclease cleaves as a dimer, one strategy to prevent off-targetcleavage events has been to design zinc finger domains that bind atadjacent 9 base pair sites.

Another class of artificial endonucleases is the engineeredmeganucleases. Engineered homing endonucleases are generated bymodifying the specificity of existing homing endonucleases. In oneapproach, variations are introduced in the amino acid sequence ofnaturally occurring homing endonucleases and then the resultantengineered homing endonucleases are screened to select functionalproteins which cleave a targeted binding site. In another approach,chimeric homing endonucleases are engineered by combining therecognition sites of two different homing endonucleases to create a newrecognition site composed of a half-site of each homing endonuclease.

The mutagenicity of the double strand DNA breaks generated by both thenaturally occurring and artificial endonucleases depend upon theprecision of DNA repair. The double strand breaks caused byendonucleases are commonly repaired through non-homologous end joining(NHEJ), which is the major DNA double-strand break repair pathway formany organisms. NHEJ is referred to as “non-homologous” because thebreak ends are ligated directly without the need for a homologoustemplate, in contrast to homologous recombination, which utilizes ahomologous sequence to guide repair. Imprecise repair through thispathway can result in mutations at the break site, such as DNA basedeletions and insertions as well as translocations and telomere fusion.When the mutations are made within the coding sequence of a gene, theycan render the gene and its subsequent protein product non-functional,creating a targeted gene disruption or “knockout” of the gene.

Double strand DNA break repair through the NHEJ pathway is often notmutagenic. The majority of endonuclease-induced breaks repaired by theNHEJ pathway involve precise re-ligation, resulting in the restorationof the original DNA sequence. This is especially true of the types ofDNA breaks created by the current endonuclease platforms available forengineering site-specificity, namely homing endonucleases(meganucleases) and zinc finger nucleases. Both of these types ofenzymes leave compatible base pair overhangs that do not requireprocessing prior to re-ligation by the NHEJ pathway. When the overhangsare compatible, NHEJ repairs the break with a high degree of accuracy.Thus, from a genome engineering standpoint, many of the cleavage eventsgenerated by the current site-specific endonuclease platforms areunproductive.

The need for additional solutions to these problems is manifest.

SUMMARY

Mutagenesis of cellular DNA can occur when a DNA cleavage event isfollowed by imprecise end joining during DNA repair. As disclosedherein, one strategy for increasing the frequency of imprecise DNArepair events is by modifying compatible overhangs generated atdouble-strand DNA breaks with an end-processing enzyme. The methods andcompositions described herein are broadly applicable and may involve anyagent of interest which generates either blunt ends or compatibleoverhangs upon cleaving double stranded DNA, for example, nucleases,ionizing radiation, such as x-rays and gamma rays, as well as drugs suchas bleomycin, cisplatin, and mitomycin C. Several embodiments disclosedherein relate to methods for coupling the generation of double-strandDNA breaks to modification of compatible overhangs generated at thecleavage site with a DNA end-processing enzyme. Several embodimentsdisclosed herein relate to methods for coupling the generation ofdouble-strand DNA breaks to modification of blunt ends generated at thecleavage site with an end-processing enzyme. Some embodiments disclosedherein relate to methods for coupling the generation of double-strandDNA breaks to cleavage of the exposed phosphodiester bonds at the DNAbreak site by an exonuclease. Some embodiments disclosed herein relateto methods for coupling the generation of double-strand DNA breaks tothe addition of DNA bases to an exposed DNA end by a non-templatepolymerase.

In yet another aspect, the methods and compositions described herein arebroadly applicable and may involve any agent of interest which generatesbreaks in a polynucleatide. Several embodiments disclosed herein relateto methods for coupling the generation of polynucleotide breaks tomodification of polynucleotide ends generated at the cleavage site withan end-processing enzyme. In some embodiments, the polynucleotide may bedouble stranded DNA, single stranded DNA, stranded RNA, single strandedRNA, double stranded DNA/RNA hybrids and synthetic polynucleotides.

Several embodiments disclosed herein relate to a strategy for increasingthe frequency of imprecise DNA repair events by modifying compatibleoverhangs generated at exonuclease-induced DNA breaks with a DNAend-processing enzyme. Several embodiments disclosed herein relate tomethods for coupling site-specific cleavage of a targeted DNA sequenceto modification of compatible overhangs generated at the cleavage sitewith a DNA end-processing enzyme. Several embodiments disclosed hereinrelate to methods for coupling site-specific cleavage of a targeted DNAsequence to modification of blunt DNA ends generated at the cleavagesite with a DNA end-processing enzyme. Some embodiments disclosed hereinrelate to methods for coupling site-specific cleavage of a targeted DNAsequence by an endonuclease to cleavage of the exposed phosphodiesterbonds at the DNA cleavage site by an exonuclease. Some embodimentsdisclosed herein relate to methods for coupling site-specific cleavageof a targeted DNA sequence by an endonuclease to the addition of DNAbases to an exposed DNA end by a non-template polymerase. Someembodiments disclosed herein relate to methods for couplingsite-specific cleavage of a targeted DNA sequence by an endonuclease toremoval of a 5′phosphate at the DNA cleavage site by a 5′-phosphatase.Some embodiments disclosed herein relate to methods for couplingsite-specific cleavage of a targeted DNA sequence by an endonuclease toremoval of a 3′ phosphate at the DNA cleavage site by a 3′ phosphatase.Further disclosed herein are fusion proteins, comprising one or moresite-specific endonuclease domains tethered to one or more DNAend-processing domains.

Non-limiting examples of endonucleases include homing endonucleases(meganucleases), zinc finger nucleases and TAL effector nucleases. Theendonucleases may comprise heterologous DNA-binding and cleavage domains(e.g., zinc finger nucleases; homing endonuclease DNA-binding domainswith heterologous cleavage domains or TAL-effector domain nucleasefusions) or, alternatively, the DNA-binding domain of anaturally-occurring nuclease may be altered to bind to a selected targetsite (e.g., a homing endonuclease that has been engineered to bind tosite different than the cognate binding site or a TAL-effector domainnuclease fusion).

Non-limiting examples of DNA end-processing enzymes include5-3′exonucleases, 3-5′exonucleases, 5-3′ alkaline exonucleases, 5′ flapendonucleases, helicases, phosphatases, hydrolases andtemplate-independent DNA polymerases. The exonucleases may compriseheterologous DNA-binding and end-processing domains (e.g., a zinc fingerand an exonuclease domain).

Several embodiments relate to co-expression of one or more endonucleases(enzymes that incise DNA at a specific internal target site) with one ormore end-processing enzymes, in order to achieve enhanced processing ofthe polynucleotide ends produced by endonuclease-mediated polynucleotidecleavage. Several embodiments relate to co-expression of one or moreendonucleases with one or more exonucleases (enzymes that catalyzes theremoval of polynucleotide bases from an exposed polynucleotide end) inorder to achieve enhanced processing of the polynucleotide ends producedby endonuclease-mediated polynucleotide cleavage. Several embodimentsrelate to co-expression of one or more endonucleases with one or morenon-templative polymerases (enzymes that catalyze the addition of DNAbases to an exposed DNA end) in order to achieve enhanced processing ofthe DNA ends produced by endonuclease-mediated DNA cleavage. Severalembodiments relate to co-expression of one or more endonucleases withone or more phosphatases that catalyze the removal of a 5′ phosphate inorder to achieve enhanced processing of the polynucleotide ends producedby endonuclease-mediated polynucleotide cleavage. Several embodimentsrelate to co-expression of one or more endonucleases with one or morephosphatases that catalyze the removal of a 3′ phosphate in order toachieve enhanced processing of the polynucleotide ends produced byendonuclease-mediated polynucleotide cleavage. In some embodiments, anendonuclease is coupled to an end-processing enzyme.

Several embodiments relate to co-expression of one or more endonucleases(enzymes that incise DNA at a specific internal target site) with one ormore DNA end-processing enzymes, in order to achieve enhanced processingof the DNA ends produced by endonuclease-mediated DNA cleavage. Severalembodiments relate to co-expression of one or more endonucleases withone or more exonucleases (enzymes that catalyzes the removal of DNAbases from an exposed DNA end) in order to achieve enhanced processingof the DNA ends produced by endonuclease-mediated DNA cleavage. Severalembodiments relate to co-expression of one or more endonucleases withone or more non-templative polymerases (enzymes that catalyze theaddition of DNA bases to an exposed DNA end) in order to achieveenhanced processing of the DNA ends produced by endonuclease-mediatedDNA cleavage. Several embodiments relate to co-expression of one or moreendonucleases with one or more phosphatases that catalyze the removal ofa 5′ phosphate in order to achieve enhanced processing of the DNA endsproduced by endonuclease-mediated DNA cleavage. Several embodimentsrelate to co-expression of one or more endonucleases with one or morephosphatases that catalyze the removal of a 3′ phosphate in order toachieve enhanced processing of the DNA ends produced byendonuclease-mediated DNA cleavage. In some embodiments, an endonucleaseis coupled to a DNA end-processing enzyme.

In one aspect, a method for improving the mutation frequency associatedwith endonuclease mediated cleavage of cellular DNA in a region ofinterest (e.g., a method for targeted disruption of genomic sequences)is provided, the method comprising: (a) selecting a sequence in theregion of interest; (b) selecting a site-specific endonuclease whichcleaves the sequence within the region of interest; and (c) deliveringone or more fusion proteins to the cell, the fusion protein(s)comprising one or more site-specific endonuclease domains and one ormore DNA end-processing domains; wherein the endonuclease domain cleavesthe DNA in the region of interest. In some embodiments, a fusion proteincan be delivered to a cell by delivering a polynucleotide encoding thefusion protein to a cell. In some embodiments the polynucleotide is DNA.In other embodiments, the polynucleotide is RNA. In some embodiments, afusion protein can be expressed in a cell by delivering a DNA vectorencoding the fusion protein to a cell, wherein the DNA vector istranscribed and the mRNA transcription product is translated to generatethe fusion protein. In some embodiments, a fusion protein can beexpressed in a cell by delivering an RNA molecule encoding the fusionprotein to the cell wherein the RNA molecule is translated to generatethe fusion protein. In some embodiments, a fusion protein may bedelivered directly to the cell.

In another aspect, a method for improving the mutation frequencyassociated with endonuclease mediated cleavage of cellular DNA in aregion of interest (e.g., a method for targeted disruption of genomicsequences) is provided, the method comprising: (a) selecting a sequencein the region of interest; (b) selecting one or more site-specificendonucleases which cleaves the sequence within the region of interest;and (c) co-expressing the one or more selected endonuclease and one ormore end-processing enzyme in the cell; wherein the endonuclease cleavesthe DNA in the region of interest and the end-processing enzyme modifiesthe DNA ends exposed by the endonuclease. The nucleases andend-processing enzymes can be expressed in a cell, e.g., by deliveringthe proteins to the cell or by delivering one or more polynucleotidesencoding the nucleases to a cell. In some embodiments, a singlepolynucleotide encodes both the one or more endonucleases and the one ormore end-processing enzymes under the control of a single promoter. Insome embodiments, one or more endonucleases and one or moreend-processing enzymes are coupled by one or more T2A “skip” peptidemotifs. In some embodiments, one or more endonucleases and one or moreend-processing enzymes are encoded by separate polynucleotides. In someembodiments, expression of the DNA end-processing enzyme precedes thatof the endonuclease.

In yet another aspect, a method for improving the mutation frequencyassociated with endonuclease mediated cleavage of cellular DNA inmultiple regions of interest (e.g., a method for targeted disruption ofmultiple genomic sequences) is provided, the method comprising: (a)selecting a first sequence in a first region of interest; (b) selectinga first site-specific endonuclease which cleaves the first sequencewithin the first region of interest; (c) selecting a second sequence ina second region of interest; (d) selecting a second site-specificendonuclease which cleaves the second sequence within the second regionof interest and (c) co-expressing the selected endonucleases and one ormore end-processing enzymes in the cell; wherein the first endonucleasecleaves the DNA in the first region of interest, the second endonucleasecleaves the DNA in the second region of interest and the one or moreend-processing enzymes modify the exposed DNA ends. The nucleases andend-processing enzyme(s) can be expressed in a cell, e.g., by deliveringthe proteins to the cell or by delivering one or more polynucleotidesencoding the nucleases and end-processing enzyme(s) to a cell. In someembodiments, a single polynucleotide encodes both the first and secondendonucleases and the one or more end-processing enzyme under thecontrol of a single promoter. In some embodiments, the endonucleases andthe end-processing enzyme(s) are coupled by one or more T2A “skip”peptide motifs. In some embodiments, the first and second regions ofinterest are in the same gene. In other embodiments, the first andsecond regions of interest are in different genes. In some embodimentsthe method further comprises co-expression of a third, fourth, fifth,sixth, seventh, eighth, ninth, and/or tenth endonuclease in the cell.

In yet another aspect, the disclosure provides a method for treating orpreventing, or inhibiting HIV infection or ameliorating a conditionassociated with HIV in a subject, the method comprising: (a)introducing, into a cell, a first nucleic acid encoding a firstpolypeptide, wherein the first polypeptide comprises: (i) a zinc fingerDNA-binding domain that is engineered to bind to a first target site inthe CCR5 gene; and (ii) a cleavage domain; and (iii) an end-processingdomain under conditions such that the polypeptide is expressed in thecell, whereby the polypeptide binds to the target site and cleaves theCCR5 gene and end-processing enzyme domain modifies the endonucleasecleavage site; and (b) introducing the cell into the subject. In certainembodiments, the cell is selected from the group consisting of ahematopoietic stem cell, a T-cell, a macrophage, a dendritic cell, andan antigen-presenting cell.

In yet another aspect, the disclosure provides a method for treating orpreventing or inhibiting HIV infection or ameliorating a conditionassociated with HIV in a subject, the method comprising: (a)introducing, into a cell, a first nucleic acid encoding a firstpolypeptide and a second polypeptide, wherein the first polypeptidecomprises: (i) a zinc finger DNA-binding domain that is engineered tobind to a first target site in the CCR5 gene; and (ii) a cleavagedomain; and the second polypeptide comprises a end-processing enzymeunder conditions such that the polypeptides are co-expressed in thecell, whereby the first polypeptide binds to the target site and cleavesthe CCR5 gene and the end-processing enzyme modifies the exposed DNAends created at the endonuclease cleavage site; and (b) introducing thecell into the subject. In certain embodiments, the cell is selected fromthe group consisting of a hematopoietic stem cell, a T-cell, amacrophage, a dendritic cell and an antigen-presenting cell.

In another aspect, the disclosure provides a method for treating orpreventing or inhibiting HIV infection or ameliorating a conditionassociated with HIV in a subject, the method comprising: (a)introducing, into a cell, a nucleic acid encoding a polypeptide, whereinthe polypeptide comprises: (i) a homing endonuclease domain that isengineered to bind to a first target site in the CCR5 gene; and (ii) aend-processing domain under conditions such that the polypeptide isexpressed in the cell, whereby the polypeptide binds to the target siteand cleaves the CCR5 gene and modifies the exposed DNA ends created atthe cleavage site; and (b) introducing the cell into the subject. Incertain embodiments, the DNA end-processing domain comprises anexonuclease.

In another aspect, the disclosure provides a method for treating orpreventing or inhibiting HIV infection or ameliorating a conditionassociated with HIV in a subject, the method comprising: (a)introducing, into a cell, a nucleic acid encoding a first polypeptideand a second polypeptide, wherein the first polypeptide comprises ahoming endonuclease that is engineered to bind to a target site in theCCR5 gene; and the second polypeptide comprises a end-processing enzymeunder conditions such that the polypeptides are co-expressed in thecell, whereby the first polypeptide binds to the target site and cleavesthe CCR5 gene and the end-processing enzyme modifies the exposed DNAends created at the endonuclease cleavage site; and (b) introducing thecell into the subject. In certain embodiments, the end-processing enzymecomprises an exonuclease. In some embodiments, the homing endonucleaseand the end-processing enzyme are coupled by one or more T2A “skip”peptide motifs.

In yet another aspect, the disclosure provides a method for treating orpreventing or inhibiting HIV infection or ameliorating a conditionassociated with HIV in a subject, the method comprising: (a)introducing, into a cell, a first nucleic acid encoding a firstpolypeptide, wherein the first polypeptide comprises: a homingendonuclease that is engineered to bind to a first target site in theCCR5 gene; and (b) introducing, into the cell, a second nucleic acidencoding a second polypeptide, wherein the second polypeptide comprises:a end-processing enzyme; under conditions such that the polypeptides areexpressed in the cell, whereby the homing endonuclease binds to thetarget site and cleaves the CCR5 gene and the end-processing enzymemodifies the exposed DNA ends created at the endonuclease cleavage site;and (b) introducing the cell into the subject. In certain embodiments,the end-processing enzyme comprises an exonuclease. In some embodiments,expression of the end-processing enzyme precedes that of theendonuclease.

In another aspect, the disclosure provides a method for treating orpreventing or inhibiting hyper IGE syndrome or ameliorating a conditionassociated with hyper IGE syndrome a subject, the method comprising: (a)introducing, into one or more cells, a nucleic acid encoding apolypeptide, wherein the polypeptide comprises: (i) a homingendonuclease domain that is engineered to bind to a first target site inthe Stat3 gene; and (ii) a end-processing domain under conditions suchthat the polypeptide is expressed in the cell, whereby the polypeptidebinds to the target site and cleaves the Stat3 gene and modifies theexposed DNA ends created at the endonuclease cleavage site. In certainembodiments, the end-processing enzyme domain comprises an exonuclease.

In yet another aspect, the disclosure provides a method for treating orpreventing or inhibiting hyper IGE syndrome or ameliorating a conditionassociated with hyper IGE syndrome a subject, the method comprising: (a)introducing, into a cell, a first nucleic acid encoding a firstpolypeptide, wherein the first polypeptide comprises: a homingendonuclease that is engineered to bind to a first target site in theSTAT3 gene; and (b) introducing, into the cell, a second nucleic acidencoding a second polypeptide, wherein the second polypeptide comprises:a end-processing enzyme; under conditions such that the polypeptides areexpressed in the cell, whereby the homing endonuclease binds to thetarget site and cleaves the STAT3 gene and the end-processing enzymemodifies the exposed DNA ends created at the endonuclease cleavage site.In certain embodiments, the end-processing enzyme comprises anexonuclease. In some embodiments, the expression of the end-processingenzyme precedes that of the endonuclease.

In yet another aspect, the disclosure provides a method for treating orpreventing or inhibiting hyper IGE syndrome or ameliorating a conditionassociated with hyper IGE syndrome a subject, the method comprising: (a)introducing, into a cell, a nucleic acid encoding a first polypeptideand a second polypeptide, wherein the first polypeptide comprises ahoming endonuclease that is engineered to bind to a first target site inthe STAT3 gene and the second polypeptide comprises a end-processingenzyme; under conditions such that the polypeptides are co-expressed inthe cell, whereby the homing endonuclease binds to the target site andcleaves the STAT3 gene and the end-processing enzyme modifies theexposed DNA ends created at the endonuclease cleavage site. In certainembodiments, the end-processing enzyme comprises an exonuclease. In someembodiments, the homing endonuclease and the end-processing enzyme arecoupled by one or more T2A “skip” peptide motifs.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a schematic of the Traffic Light Reporter system (TLR) formeasuring the effectiveness of exonuclease induced gene disruption.mCherry positive cells represent a proportion of the total cells thathave undergone gene disruption. FIGS. 1B-1H show schematicrepresentations of expression vectors for delivery of endonucleases andDNA end-processing enzymes.

FIG. 2A shows representative flow plots of HEK293 cells harboringTraffic light Reporter transfected with expression vectors encodingSceD44A-IRES-BFP, SceD44A-T2A-Trex2-IRES-BFP, I-SceI-IRES-BFP, andI-SceI-T2A-Trex2-IRES-BFP. SceD44A corresponds to an inactive mutantform of I-SceI. FIG. 2B shows quantification of gene disruption in threeindependent transfections of the vectors indicated in FIG. 2A. Errorbars represent standard error of the mean (SEM), and p-values (with *representing p<0.05, ** p<0.005, and *** p<0.0005) were calculated usingthe Student's two-tailed unpaired t-test to compare the samplesindicated in this and all subsequent figures.

FIG. 3A shows representative flow plots of HEK293 cells harboringTraffic light Reporter transfected with expression vectors encodingI-SceI-IRES-BFP, I-SceI-T2A-Trex2-BFP, or I-SceI-G4S-Trex2-IRES BFP.FIG. 3B shows an anti-HA western blot demonstrating equal expression ofendonucleases, and stability of the (HA-)I-SceI, (HA-)I-SceI-T2A and(HA-)I-SceI-G4S-Trex2 proteins from FIG. 3A. FIG. 3C is a licor westernblot showing size and stability of the HA-tagged I-SceI in indicatedHEK293T lysates.

FIG. 4A shows gating analysis of HEK293 cells harboring Traffic LightReporter transfected with I-SceI-IRES-BFP. FIG. 4B shows a gatinganalysis of HEK293 cells harboring Traffic Light Reporter transfectedwith I-SceI-T2A-Trex2-IRES-BFP expression vectors.

FIG. 5A shows an I-SceI restriction digest of amplicons flanking theI-SceI target site from HEK293 cells harboring traffic light reportersorted by BFP expression levels follow transfection with expressionconstructs as indicated in FIG. 4A and FIG. 4B. FIG. 5B showsquantification of three independent experiments as described in FIG. 5A.

FIGS. 6A and 6B shows the results of DNA sequencing of ampliconssurrounding the I-SceI target site in HEK293 Traffic Light Reportercells treated with I-SceI-IRES-BFP or I-SceI-T2A-Trex2-IRES-BFP.

FIG. 7 shows a graph scoring observed mutations (deletions are negative,insertions are positive) at the I-SceI target site followingtransfection of HEK293 Traffic Light Reporter cells with I-SceI-IRES-BFPor I-SceI-T2A-Trex2-IRES-BFP as described in FIG. 5.

FIG. 8A shows a kinetic time course analysis demonstrating transientexpression of I-SceI-T2A-Trex2-IRES-BFP after transfection into HEK293cells harboring Traffic Light Reporter. The constructs shown are taggedto BFP by an IRES sequence downstream of either I-SceI or Trex2. FIG. 8Bshows a graph quantifying 3 experiments of HEK293T cells transfectedwith the vectors indicated in FIG. 8A, analyzed at the indicatedtime-points. Cherry indicates gene disruption rates observed intransfected cells. FIG. 9A shows an I-SceI restriction digest ofamplicons from primary murine embryonic fibroblasts spanning an I-SceItarget site 72 hours post transduction with I-SceI-IRES BFP orI-SceI-T2A-Trex2-IRES-BFP. FIG. 9B shows a graph quantifying cleavagesite disruption in 2 independent experiments. FIG. 9C shows an I-SceIrestriction digest of amplicons from lineage depleted bone marrowspanning an I-SceI target site 72 hours post transduction withI-SceI-IRES BFP or I-SceI-T2A-Trex2-IRES-BFP. FIG. 9D showsquantification of bands from FIG. 9C.

FIG. 10 shows a graph quantifying gene disruption rates of severaldifferent homing endonucleases with and without Trex2 exonuclease asmeasured by HEK293 cells harboring Traffic Light Reporters withrespective target sites for the indicated homing endonucleases.

FIG. 11A shows representative flow plots and targets sites of HEK293Traffic Light Reporter cells following transfection with a homingendonuclease with and without Trex2 and a zinc finger nuclease with andwithout Trex2. FIG. 11B shows a graph of an independent experimentexamining cleavage site mutation for I-SceI and Zinc Finger Nuclease inthe presence and absence of Trex2. FIG. 11C shows a graph of HEK293Traffic Light Reporter cells following co-transfection of an HE withTrex2 or a TALEN with Trex2.

FIG. 12A shows representative flow plots of HEK293 cells harboringTraffic Light Reporters with an I-AniI target site followingtransfection with either I-AniI-IRES-BFP, I-AniI-T2A-Trex2-IRES-BFP,I-AniIY2-IRES-BFP, I-AniIY2-T2A-Trex2-IRES-BFP. FIG. 12B shows a graphquantitating 3 independent experiments as performed in FIG. 12A.

FIG. 13 shows graph depicting cell cycle analysis of murine embryonicfibroblasts transduced with Mock, I-SceI-IRES-BFP, orI-SceI-T2A-Trex2-IRES-BFP viruses.

FIG. 14 shows a graph depicting maintenance of BFP expression in cellstransduced with an integrating lentivirus containing I-SceID44A-IRES-BFPor I-SceID44A-T2A-Trex2-IRES-BFP.

FIG. 15A shows a graph measuring human CD34+ hematopietic stem cellsurvival when transduced with I-SceID44A-IRES-BFP orI-SceID44A-T2A-Trex2-IRES-BFP and challenged with Mitomycin C. FIG. 15Bshows a graph measuring human CD34+ hematopietic stem cell survival whentransduced with I-SceID44A-IRES-BFP or I-SceID44A-T2A-Trex2-IRES-BFP andchallenged with camptothecin. FIG. 15C shows a graph measuring humanCD34+ hematopietic stem cell survival when transduced withI-SceID44A-IRES-BFP or I-SceID44A-T2A-Trex2-IRES-BFP and challenged withionizing radiation.

FIG. 16A shows a graph measuring murine embryonic fibroblast cellsurvival when transduced with I-SceID44A-IRES-BFP orI-SceID44A-T2A-Trex2-IRES-BFP and challenged with Mitomycin C. FIG. 16Bshows a graph measuring murine embryonic fibroblast cell survival whentransduced with I-SceID44A-IRES-BFP or I-SceID44A-T2A-Trex2-IRES-BFP andchallenged with camptothecin.

FIG. 17A ₁ and FIG. 17A ₂ show representative flow plots of HEK293Traffic Light Reporter cells following co-transfection ofI-SceI-IRES-BFP and an expression plasmid coding for the indicatedend-processing enzyme. FIG. 17B shows a graph quantifying 3 independentexperiments as performed in FIG. 17A ₁ and FIG. 17A ₂.

FIG. 18A shows representative flow plots of a gating analysis ofI-SceI-IRES-BFP co-transfected with ARTEMIS expression plasmid asindicated in FIG. 17A ₁ and FIG. 17A ₂. FIG. 18B shows a graphquantifying gating analysis of several end-processing enzymes from 3independent experiments as indicated in FIG. 18A ₁ and FIG. 17A ₂.

FIG. 19A shows a graph of HEK293 Traffic Light Reporter cells followingco-transfection with a zinc finger nuclease and the indicatedend-processing enzyme expression plasmid. FIG. 19B shows a graph ofHEK293 Traffic Light Reporter cells following co-transfection with aTALEN and the indicated end-processing enzyme expression plasmid.

FIGS. 20A and 20B show a comparison of expression levels and genedisruption rates between integrating lentivirus and integrase deficientlentivirus from I-SceI with and without exonuclease coupling on HEK293Traffic Light reporter cells.

FIG. 21A shows live cell image of cells 72 hrs post mock transfection ortransfection with an expression vectors encoding I-SceI-IRES-BFP orI-SceI-T2A-Trex2-IRES-BFP. FIG. 21B shows a graph depicting maintenanceof BFP expression in cells transduced with an integrating lentiviruscontaining BFP alone (no Trex2) or Trex2-BFP.

DETAILED DESCRIPTION Definitions

In the description that follows, a number of terms are used extensively.The following definitions are provided to facilitate understanding ofthe present embodiments.

As used herein, “a” or “an” may mean one or more than one.

As used herein, the term “about” indicates that a value includes theinherent variation of error for the method being employed to determine avalue, or the variation that exists among experiments.

As used herein, “nucleic acid” or “nucleic acid molecule” refers topolynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid(RNA), oligonucleotides, fragments generated by the polymerase chainreaction (PCR), and fragments generated by any of ligation, scission,endonuclease action, and exonuclease action. Nucleic acid molecules canbe composed of monomers that are naturally-occurring nucleotides (suchas DNA and RNA), or analogs of naturally-occurring nucleotides (e.g.,enantiomeric forms of naturally-occurring nucleotides), or a combinationof both. Modified nucleotides can have alterations in sugar moietiesand/or in pyrimidine or purine base moieties. Sugar modificationsinclude, for example, replacement of one or more hydroxyl groups withhalogens, alkyl groups, amines, and azido groups, or sugars can befunctionalized as ethers or esters. Moreover, the entire sugar moietycan be replaced with sterically and electronically similar structures,such as aza-sugars and carbocyclic sugar analogs. Examples ofmodifications in a base moiety include alkylated purines andpyrimidines, acylated purines or pyrimidines, or other well-knownheterocyclic substitutes. Nucleic acid monomers can be linked byphosphodiester bonds or analogs of such linkages. Analogs ofphosphodiester linkages include phosphorothioate, phosphorodithioate,phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate,phosphoranilidate, phosphoramidate, and the like. The term “nucleic acidmolecule” also includes so-called “peptide nucleic acids,” whichcomprise naturally-occurring or modified nucleic acid bases attached toa polyamide backbone. Nucleic acids can be either single stranded ordouble stranded.

The term “contig” denotes a nucleic acid molecule that has a contiguousstretch of identical or complementary sequence to another nucleic acidmolecule. Contiguous sequences are said to “overlap” a given stretch ofa nucleic acid molecule either in their entirety or along a partialstretch of the nucleic acid molecule.

The term “degenerate nucleotide sequence” denotes a sequence ofnucleotides that includes one or more degenerate codons as compared to areference nucleic acid molecule that encodes a polypeptide. Degeneratecodons contain different triplets of nucleotides, but encode the sameamino acid residue (e.g., GAU and GAC triplets each encode Asp). It willbe understood that, as a result of the degeneracy of the genetic code, amultitude of nucleotide sequences encoding a given protein such as anendonuclease, end-processing enzyme, or endonuclease/end-processingenzyme fusion protein of the present embodiments may be produced.

The term “complementary to” means that the complementary sequence ishomologous to all or a portion of a reference polynucleotide sequence.For illustration, the nucleotide sequence “CATTAG” corresponds to areference sequence “CATTAG” and is complementary to a reference sequence“GTAATC.”

The term “structural gene” refers to a nucleic acid molecule that istranscribed into messenger RNA (mRNA), which is then translated into asequence of amino acids characteristic of a specific polypeptide.

An “isolated nucleic acid molecule” is a nucleic acid molecule that isnot integrated in the genomic DNA of an organism. For example, a DNAmolecule that encodes a growth factor that has been separated from thegenomic DNA of a cell is an isolated DNA molecule. Another non-limitingexample of an isolated nucleic acid molecule is a chemically-synthesizednucleic acid molecule that is not integrated in the genome of anorganism. A nucleic acid molecule that has been isolated from aparticular species is smaller than the complete DNA molecule of achromosome from that species.

“Complementary DNA (cDNA)” is a single-stranded DNA molecule that isformed from an mRNA template by the enzyme reverse transcriptase.Typically, a primer complementary to portions of mRNA is employed forthe initiation of reverse transcription. Those skilled in the art mayalso use the term “cDNA” to refer to a double-stranded DNA moleculeconsisting of such a single-stranded DNA molecule and its complementaryDNA strand. The term “cDNA” may also refer to a clone of a cDNA moleculesynthesized from an RNA template.

A “promoter” is a nucleotide sequence that directs the transcription ofa structural gene. In some embodiments, a promoter is located in the 5′non-coding region of a gene, proximal to the transcriptional start siteof a structural gene. Sequence elements within promoters that functionin the initiation of transcription are often characterized by consensusnucleotide sequences. These promoter elements include RNA polymerasebinding sites, TATA sequences, CAAT sequences, differentiation-specificelements (DSEs; McGehee et al., Mol. Endocrinol. 7:551 (1993)), cyclicAMP response elements (CREs), serum response elements (SREs; Treisman,Seminars in Cancer Biol. 1:47 (1990)), glucocorticoid response elements(GREs), and binding sites for other transcription factors, such asCRE/ATF (O'Reilly et al., J. Biol. Chem. 267:19938 (1992)), AP2 (Ye etal., J. Biol. Chem. 269:25728 (1994)), SP1, cAMP response elementbinding protein (CREB; Loeken, Gene Expr. 3:253 (1993)) and octamerfactors (see, in general, Watson et al., eds., Molecular Biology of theGene, 4th ed. (The Benjamin/Cummings Publishing Company, Inc. 1987), andLemaigre and Rousseau, Biochem. J. 303:1 (1994)). As used herein, apromoter may be constitutively active, repressible or inducible. If apromoter is an inducible promoter, then the rate of transcriptionincreases in response to an inducing agent. In contrast, the rate oftranscription is not regulated by an inducing agent if the promoter is aconstitutive promoter. Repressible promoters are also known.

A “core promoter” contains essential nucleotide sequences for promoterfunction, including the TATA box and start of transcription. By thisdefinition, a core promoter may or may not have detectable activity inthe absence of specific sequences that may enhance the activity orconfer tissue specific activity.

A “regulatory element” is a nucleotide sequence that modulates theactivity of a core promoter. For example, a regulatory element maycontain a nucleotide sequence that binds with cellular factors enablingtranscription exclusively or preferentially in particular cells,tissues, or organelles. These types of regulatory elements are normallyassociated with genes that are expressed in a “cell-specific,”“tissue-specific,” or “organelle-specific” manner.

An “enhancer” is a type of regulatory element that can increase theefficiency of transcription, regardless of the distance or orientationof the enhancer relative to the start site of transcription.

“Heterologous DNA” refers to a DNA molecule, or a population of DNAmolecules, that does not exist naturally within a given host cell. DNAmolecules heterologous to a particular host cell may contain DNA derivedfrom the host cell species (e.g., endogenous DNA) so long as that hostDNA is combined with non-host DNA (e.g., exogenous DNA). For example, aDNA molecule containing a non-host DNA segment encoding a polypeptideoperably linked to a host DNA segment comprising a transcriptionpromoter is considered to be a heterologous DNA molecule. Conversely, aheterologous DNA molecule can comprise an endogenous gene operablylinked with an exogenous promoter. As another illustration, a DNAmolecule comprising a gene derived from a wild-type cell is consideredto be heterologous DNA if that DNA molecule is introduced into a mutantcell that lacks the wild-type gene.

A “polypeptide” is a polymer of amino acid residues joined by peptidebonds, whether produced naturally or synthetically. Polypeptides of lessthan about 10 amino acid residues are commonly referred to as“peptides.”

A “protein” is a macromolecule comprising one or more polypeptidechains. A protein may also comprise non-peptide components, such ascarbohydrate groups. Carbohydrates and other non-peptide substituentsmay be added to a protein by the cell in which the protein is produced,and will vary with the type of cell. Proteins are defined herein interms of their amino acid backbone structures; substituents such ascarbohydrate groups are generally not specified, but may be presentnonetheless.

A peptide or polypeptide encoded by a non-host DNA molecule is a“heterologous” peptide or polypeptide.

An “integrated genetic element” is a segment of DNA that has beenincorporated into a chromosome of a host cell after that element isintroduced into the cell through human manipulation. Within the presentembodiments, integrated genetic elements are most commonly derived fromlinearized plasmids that are introduced into the cells byelectroporation or other techniques. Integrated genetic elements arepassed from the original host cell to its progeny.

A “cloning vector” is a nucleic acid molecule, such as a plasmid,cosmid, plastome, or bacteriophage that has the capability ofreplicating autonomously in a host cell. Cloning vectors typicallycontain one or a small number of restriction endonuclease recognitionsites that allow insertion of a nucleic acid molecule in a determinablefashion without loss of an essential biological function of the vector,as well as nucleotide sequences encoding a marker gene that is suitablefor use in the identification and selection of cells transduced with thecloning vector. Marker genes typically include genes that providetetracycline resistance or ampicillin resistance.

An “expression vector” is a nucleic acid molecule encoding a gene thatis expressed in a host cell. Typically, an expression vector comprises atranscription promoter, a gene, and a transcription terminator. Geneexpression is usually placed under the control of a promoter, and such agene is said to be “operably linked to” the promoter. Similarly, aregulatory element and a core promoter are operably linked if theregulatory element modulates the activity of the core promoter.

As used herein, “transient transfection” refers to the introduction ofexogenous nucleic acid(s) into a host cell by a method that does notgenerally result in the integration of the exogenous nucleic into thegenome of the transiently transfected host cell.

By the term “host cell” is meant a cell that contains one or morenucleases, for example endonucleases, end-processing enzymes, and/orendonuclease/end-processing enzyme fusion proteins encompassed by thepresent embodiments or a vector encoding the same that supports thereplication, and/or transcription or transcription and translation(expression) of one or more nucleases, for example endonucleases,end-processing enzymes, and/or endonuclease/end-processing enzyme fusionproteins. Host cells for use in the present invention can be prokaryoticcells or eukaryotic cells. Examples of prokaryotic host cells include,but are not limited to E. coli, nitrogen fixing bacteria, Staphylococcusaureus, Staphylococcus albus, lactobacillus acidophilus, Bacillusanthracis, Bacillus subtilis, Bacillus thuringiensis, Clostridiumtetani, Clostridium botulinum, Streptococcus mutans, Streptococcuspneumoniae, mycoplasmas, and cyanobacteria. Examples of eukaryotic hostcells include, but are not limited to, protozoa, fungi, algae, plant,insect, amphibian, avian and mammalian cells.

“Integrative transformants” are recombinant host cells, in whichheterologous DNA has become integrated into the genomic DNA of thecells.

An “isolated polypeptide” is a polypeptide that is essentially free fromcontaminating cellular components, such as carbohydrate, lipid, or otherproteinaceous impurities associated with the polypeptide in nature.Typically, a preparation of isolated polypeptide contains thepolypeptide in a highly purified form, e.g., at least about 80% pure, atleast about 90% pure, at least about 95% pure, greater than 95% pure, orgreater than 99% pure. One way to show that a particular proteinpreparation contains an isolated polypeptide is by the appearance of asingle band following sodium dodecyl sulfate (SDS)-polyacrylamide gelelectrophoresis of the protein preparation and Coomassie Brilliant Bluestaining of the gel. However, the term “isolated” does not exclude thepresence of the same polypeptide in alternative physical forms, such asdimers, or alternatively glycosylated or derivative forms.

The terms “amino-terminal” and “carboxyl-terminal” are used herein todenote positions within polypeptides. Where the context allows, theseterms are used with reference to a particular sequence or portion of apolypeptide to denote proximity or relative position. For example, acertain sequence positioned carboxyl-terminal to a reference sequencewithin a polypeptide is located proximal to the carboxyl terminus of thereference sequence, but is not necessarily at the carboxyl terminus ofthe complete polypeptide.

The term “gene expression” refers to the biosynthesis of a gene product.For example, in the case of a structural gene, gene expression involvestranscription of the structural gene into mRNA and the translation ofmRNA into one or more polypeptides.

The term “endonuclease” refers to enzymes that cleave the phosphodiesterbond within a polynucleotide chain. The polynucleotide may bedouble-stranded DNA (dsDNA), single-stranded DNA (ssDNA), RNA,double-stranded hybrids of DNA and RNA, and synthetic DNA (for example,containing bases other than A, C, G, and T). An endonuclease may cut apolynucleotide symmetrically, leaving “blunt” ends, or in positions thatare not directly opposing, creating overhangs, which may be referred toas “sticky ends.” The methods and compositions described herein may beapplied to cleavage sites generated by endonucleases.

The term “homing endonuclease” refers to double stranded DNases thathave large, asymmetric recognition sites (12-40 base pairs). Homingendonuclease recognition sites are extremely rare. For example, an 18base pair recognition sequence will occur only once in every 7×10¹⁰ basepairs of random sequence. This is equivalent to only one site in 20mammalian-sized genomes. Unlike standard restriction endonucleases,however, homing endonucleases tolerate some sequence degeneracy withintheir recognition sequence. As a result, their observed sequencespecificity is typically in the range of 10-12 base pairs. Although thecleavage specificity of most homing endonucleases is not absolute withrespect to their recognition sites, the sites are of sufficient lengththat a single cleavage event per mammalian-sized genome can be obtainedby expressing a homing endonuclease in a cell containing a single copyof its recognition site. Examples of homing endonucleases include, butare not limited to, I-AniI, I-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV,I-CsmI, I-PanI, I-PanII, I-PanMI, I-SceII, I-PpoI, I-SceIII, I-CreI,I-LtrI, I-GpiI, I-GZeI, I-OnuI, I-HjeMI, I-TevI, I-TevII, and I-TevIII.Their recognition sequences are known. The specificity of homingendonucleases and meganucleases can be engineered to bind non-naturaltarget sites. See, for example, Chevalier et al. (2002) Molec. Cell10:895-905; Epinat et al. (2003) Nucleic Acids Res. 31:2952-2962;Ashworth et al. (2006) Nature 441:656-659; Paques et al. (2007) CurrentGene Therapy 7:49-66. The methods and compositions described herein maybe applied to cleavage sites generated by homing endonucleases.

The term “TAL effector nuclease” (TALEN) refers to a nuclease comprisinga TAL-effector domain fused to a nuclease domain. TAL-effector DNAbinding domains, isolated from the plant pathogen Xanthomonas have beendescribed (see Boch et al., (2009) Science 29 Oct. 2009(10.1126/science.117881) and Moscou and Bogdanove, (2009) Science 29Oct. 2009 (10.1126/science.1178817)). These DNA binding domains may beengineered to bind to a desired target and fused to a nuclease domain,such as the Fok1 nuclease domain, to derive a TAL effectordomain-nuclease fusion protein. The methods and compositions describedherein may be applied to cleavage sites generated by TAL effectornucleases.

The term “Zinc-finger nuclease” (ZFN) refers to artificial restrictionenzymes generated by fusing a zinc finger DNA-binding domain to aDNA-cleavage domain. Zinc finger domains can be engineered to bind to adesired target site. In some embodiments, the cleavage domain comprisesthe non-specific cleavage domain of FokI. In other embodiments, thecleavage domain comprises all or an active portion of another nuclease.In some embodiments, the cleavage domain may comprise Trex2 or an activefragment thereof. The methods and compositions described herein may beapplied to cleavage sites generated by zinc-finger nucleases

The term “end-processing enzyme” refers to an enzyme that modifies theexposed ends of a polynucleotide chain. The polynucleotide may bedouble-stranded DNA (dsDNA), single-stranded DNA (ssDNA), RNA,double-stranded hybrids of DNA and RNA, and synthetic DNA (for example,containing bases other than A, C, G, and T). An end-processing enzymemay modify exposed polynucleotide chain ends by adding one or morenucleotides, removing one or more nucleotides, removing or modifying aphosphate group and/or removing or modifying a hydroxyl group. Aend-processing enzyme may modify may modify ends at endonuclease cutsites or at ends generated by other chemical or mechanical means, suchas shearing (for example by passing through fine-gauge needle, heating,sonicating, mini bead tumbling, and nebulizing), ionizing radiation,ultraviolet radiation, oxygen radicals, chemical hydrolosis andchemotherapy agents.

The term “DNA end-processing enzyme” refers to an enzyme that modifiesthe exposed ends of DNA. A DNA end-processing enzyme may modify bluntends or staggered ends (ends with 5′ or 3′ overhangs). A DNAend-processing enzyme may modify single stranded or double stranded DNA.A DNA end-processing enzyme may modify ends at endonuclease cut sites orat ends generated by other chemical or mechanical means, such asshearing (for example by passing through fine-gauge needle, heating,sonicating, mini bead tumbling, and nebulizing), ionizing radiation,ultraviolet radiation, oxygen radicals, chemical hydrolosis andchemotherapy agents. DNA end-processing enzyme may modify exposed DNAends by adding one or more nucleotides, removing one or morenucleotides, removing or modifying a phosphate group and/or removing ormodifying a hydroxyl group. Non-limiting examples of types of DNAend-processing enzymes include 5-3′ exonucleases, 5-3′ alkalineexonucleases, 3-5′ exonucleases, 5′ flap endonucleases, helicases,phosphatases, hydrolases and template-independent DNA polymerases.Examples of DNA end-processing enzymes include, but are not limited to,Trex2, Trex1, Trex1 without transmembrane domain, Apollo, Artemis, DNA2,Exo1, ExoT, ExoIII, Fen1, Fan1, MreII, Rad2, Rad9, TdT (terminaldeoxynucleotidyl transferase), PNKP, RecE, RecJ, RecQ, Lambdaexonuclease, Sox, Vaccinia DNA polymerase, exonuclease I, exonucleaseIII, exonuclease VII, NDK1, NDK5, NDK7, NDK8, WRN, T7-exonuclease Gene6, avian myeloblastosis virus integration protein (IN), Bloom, AntarticPhophatase, Alkaline Phosphatase, Poly nucleotide Kinase (PNK), ApeI,Mung Bean nuclease, Hex1, TTRAP (TDP2), Sgs1, Sae2, CtIP, Pol mu, Pollambda, MUS81, EME1, EME2, SLX1, SLX4 and UL-12. Many DNA end-processingenzymes are highly conserved throughout evolution, and thus likely tofunction in several different species. Further, homologues of DNAend-processing enzymes may be readily identifiable in organisms ofbiotechnological interest, including plants, animals, and algae.Contemplated herein are methods of modifying DNA end-processing enzymesto optimize activity or processivity.

The term “exonuclease” refers to enzymes that cleave phosphodiesterbonds at the end of a polynucleotide chain via a hydrolyzing reactionthat breaks phosphodiester bonds at either the 3′ or 5′ end. Thepolynucleotide may be double-stranded DNA (dsDNA), single-stranded DNA(ssDNA), RNA, double-stranded hybrids of DNA and RNA, and synthetic DNA(for example, containing bases other than A, C, G, and T). The term “5′exonuclease” refers to exonucleases that cleave the phosphodiester bondat the 5′ end. The term “3′ exonuclease” refers to exonucleases thatcleave the phosphodiester bond at the 3′ end. Exonucleases may cleavethe phosphodiester bonds at the end of a polynucleotide chain atendonuclease cut sites or at ends generated by other chemical ormechanical means, such as shearing (for example by passing throughfine-gauge needle, heating, sonicating, mini bead tumbling, andnebulizing), ionizing radiation, ultraviolet radiation, oxygen radicals,chemical hydrolosis and chemotherapy agents. Exonucleases may cleave thephosphodiester bonds at blunt ends or sticky ends. E. coli exonuclease Iand exonuclease III are two commonly used 3′-exonucleases that have3′-exonucleolytic single-strand degradation activity. Other examples of3′-exonucleases include Nucleoside diphosphate kinases (NDKs), NDK1(NM23-H1), NDK5, NDK7, and NDK8 (Yoon J-H, et al., Characterization ofthe 3′ to 5′ exonuclease activity found in human nucleoside diphosphatekinase 1 (NDK1) and several of its homologues. Biochemistry2005:44(48):15774-15786.), WRN (Ahn, B., et al., Regulation of WRNhelicase activity in human base excision repair. J. Biol. Chem. 2004,279:53465-53474) and Three prime repair exonuclease 2 (Trex2) (Mazur, D.J., Perrino, F. W., Excision of 3′ termini by the Trex1 and TREX2 3′->5′exonucleases. Characterization of the recombinant proteins. J. Biol.Chem. 2001, 276:17022-17029.). E. coli exonuclease VII andT7-exonuclease Gene 6 are two commonly used 5′-3′ exonucleases that have5% exonucleolytic single-strand degradation activity. The exonucleasecan be originated from prokaryotes, such as E. coli exonucleases, oreukaryotes, such as yeast, worm, murine, or human exonucleases.

The term “cleavage” refers to the breakage of the covalent backbone of apolynucleotide. Cleavage can be initiated by a variety of methodsincluding, but not limited to, enzymatic or chemical hydrolysis of aphosphodiester bond. Both single-stranded cleavage and double-strandedcleavage are possible, and double-stranded cleavage can occur as aresult of two distinct single-stranded cleavage events. Double strandedDNA, RNA, or DNA/RNA hybrid cleavage can result in the production ofeither blunt ends or staggered ends.

The terms “target site” or “target sequence” refers to a nucleic acidsequence that defines a portion of a nucleic acid to which a bindingmolecule will bind, provided sufficient conditions for binding exist.For example, the target sites for several homing endonucleases are shownin Table 1.

TABLE 1 Examples of Homing Endonucleases and their Target Sites. HomingEndo- nucleases Target I-SceI TAGGGATAACAGGGTAAT (SEQ ID No. 1) I-LtrIAATGCTCCTATACGACGT TTAG (SEQ ID No. 2) I-GpiI TTTTCCTGTATATGACTT AAAT(SEQ ID No. 3) I-GzeI GCCCCTCATAACCCGTAT CAAG (SEQ ID No. 4) I-xMpeMITAGATAACCATAAGTGCT AAT (SEQ ID No.5) I-PanMI GCTCCTCATAATCCTTAT CAAG(SEQ ID No. 6) I-CreI TCAAAACGTCGTGAGACA GTTTGG (SEQ ID No. 7) I-OnuITTTCCACTTATTCAACCT TTTA (SEQ ID No. 8) I-HjeMI TTGAGGAGGTTTCTCTGT TAAT(SEQ ID No. 9) I-AniI TGAGGAGGTTTCTCTGTA AA (SEQ ID No. 10)

The term “fusion protein” indicates that the protein includespolypeptide components derived from more than one parental protein orpolypeptide. Typically, a fusion protein is expressed from a fusion genein which a nucleotide sequence encoding a polypeptide sequence from oneprotein is appended in frame with, and optionally separated by a linkerfrom, a nucleotide sequence encoding a polypeptide sequence from adifferent protein. The fusion gene can then be expressed by a host cellas a single protein. A fusion protein can comprise at least part of onepolypeptide fused with another polypeptide. In some embodiments, afusion protein can comprise at least a part of one polypeptide fusedwith at least a part of the same polypeptide. One example of a fusionprotein is monomorized Trex2 (at least a part of Trex2 fused to at leasta part of Trex2).

The term “endonuclease/end-processing enzyme fusion protein” or “fusionprotein having endonuclease and end-processing activity” refers to anenzyme, which has an endonuclease catalytic domain and an end-processingcatalytic domain and exhibits endonuclease and end-processing activity.

A “domain” of a protein is any portion of the entire protein, up to andincluding the complete protein, but typically comprising less than thecomplete protein. A domain can, but need not, fold independently of therest of the protein chain and/or be correlated with a particularbiological, biochemical, or structural function or location (e.g., anendonuclease domain, a polynucleotide binding domain, such as aDNA-binding domain, or an end-processing domain).

“Prokaryotic” cells lack a true nuclease. Examples of prokaryotic cellsare bacteria (e.g., cyanobacteria, Lactobacillus acidophilus,Nitrogen-Fixing Bacteria, Helicobacter pylori, Bifidobacterium,Staphylococcus aureus, Bacillus anthrax, Clostridium tetani,Streptococcus pyogenes, Staphylococcus pneumoniae, Klebsiella pneumoniaeand Escherichia coli) and archaea (e.g., Crenarchaeota, Euryarchaeota,and Korarchaeota).

“Eukaryotic” cells include, but are not limited to, algae cells, fungalcells (such as yeast), plant cells, animal cells, mammalian cells, andhuman cells (e.g., T-cells).

“Plant” cells include, but are not limited to, cells of monocotyledonous(monocots) or dicotyledonous (dicots) plants. Non-limiting examples ofmonocots include cereal plants such as maize, rice, barley, oats, wheat,sorghum, rye, sugarcane, pineapple, onion, banana, and coconut.Non-limiting examples of dicots include tobacco, tomato, sunflower,cotton, sugarbeet, potato, lettuce, melon, soybean, canola (rapeseed),and alfalfa. Plant cells may be from any part of the plant and/or fromany stage of plant development.

“Algae” are predominantly aquatic organisms that carry outoxygen-evolving photosynthesis but lack specialized water-conducting andfood-conducting tissues. Algae may be unicellular or multicellular.Algae may be adapted to live in salt water, fresh water and on land.Example of algae include, but are not limited to, diatoms, chlorophyta(for example, volvox, spirogyra), euglenophyta, dinoflagellata,chrysophyta, phaephyta (for example, fucus, kelp, sargassum), andrhodophyta (for example, lemanae).

The term “subject” as used herein includes all members of the animalkingdom including non-human primates and humans.

Overview

Several embodiments described herein relate to a method of improving therate of gene disruptions caused by imprecise repair of DNA double-strandbreaks. In some embodiments, DNA end-processing enzymes are provided toenhance the rate of gene disruption. Some aspects of the presentembodiments include, without limitation, enhanced rates of DNAend-processing enzyme-mediated processing of DNA ends at the site of adouble-strand break.

Targeted DNA double-strand breaks introduced by rare-cleavingendonucleases can be harnessed for gene disruption applications indiverse cell types by engaging non-homologous end joining DNA repairpathways. However, endonucleases create chemically clean breaks that areoften subject to precise repair, limiting the efficiency of targetedgene disruption. Several embodiments described herein relate to a methodof improving the rate of targeted gene disruptions caused by impreciserepair of endonuclease-induced site-specific DNA double-strand breaks.In some embodiments, site specific endonucleases are coupled withend-processing enzymes to enhance the rate of targeted gene disruption.Coupling may be, for example, physical, spatial, and/or temporal.

Some aspects of the present embodiments include, without limitation,enhanced rates of end-processing enzyme-mediated processing ofendonuclease-produced DNA ends, leading to enhanced targeted genedisruption at the genomic target site. Using this strategy, embodimentsdescribed herein show over 25 fold increased endonuclease-induceddisruption rates. Certain embodiments described herein can achievecomplete knockout of a target gene within a population. This technologyfurther has the potential to dramatically increase the utility ofrare-cleaving endonucleases for genetic knockout applications. Improvingthe mutation rate associated with endonucleases facilitates endonucleaseengineering, as enzymes with different levels of activity can beutilized. In some embodiments, endo-end-processor coupling is usedmodify DNA ends for endonuclease-induced genome engineering. In someembodiments, expression of exonucleases capable of processive 5′ endresection coupled with manipulation of the DNA repair environment can beused to enhance homologous recombination-mediated gene targeting.

Not to be bound by any particular theory, the resolution of adouble-strand DNA breaks by “error-prone” non-homologous end-joining(NHEJ) can be harnessed to create targeted disruptions and geneticknockouts, as the NHEJ process can result in insertions and deletions atthe site of the break. NHEJ is mediated by several sub-pathways, each ofwhich has distinct mutational consequences. The classical NHEJ pathway(cNHEJ) requires the KU/DNA-PKcs/Lig4/XRCC4 complex, and ligates endsback together with minimal processing. As the DNA breaks created bydesigner endonuclease platforms (zinc-finger nucleases (ZFNs), TALeffector nucleases (TALENs), and homing endonucleases (HEs)) all leavechemically clean, compatible overhang breaks that do not requireprocessing prior to ligation, they are excellent substrates for preciserepair by the cNHEJ pathway. In the absence or failure of the classicalNHEJ pathway to resolve a break, alternative NHEJ pathways (altNHEJ) cansubstitute: however, these pathways are considerably more mutagenic.

Not to be bound by any particular theory, modification of DNAdouble-strand breaks by end-processing enzymes may bias repair towardsan altNHEJ pathway. Further, different subsets of end-processing enzymesmay enhance disruption by different mechanisms. For example, Trex2, anexonuclease that specifically hydrolyzes the phosphodiester bonds whichare exposed at 3′ overhangs, biases repair at break sites towardmutagenic deletion. By contrast, terminal deoxynucleotidyl transferase(TdT), a non-templative polymerase, is expected to bias repair at breaksites toward mutagenic insertions by promoting the addition ofnucleotide bases to alter DNA ends prior to ligation. Accordingly, oneof skill in the art may use end-processing enzymes with differentactivities to provide for a desired engineering outcome. Further one ofskill in the art may use synergy between different end-processingenzymes to achieve maximal or unique types of knockout effects.

Several embodiments described herein couple DNA breaks created byendonucleases with end-processing enzymes is a robust way to improve therates of targeted disruption in a variety of cell types and species,without associated toxicity to the host. This is an important advance atleast because: 1) Double-strand breaks (DSBs) trigger cell cyclecheckpoints to arrest division until the break has been resolved; in thecase of a “persistent break” (a repetitive cycle of cleaving and preciserepair), cells may arrest indefinitely, leading to apoptosis. 2)Engineering applications often utilize transient delivery of anendonuclease, providing only a short window in which enzymeconcentration is sufficient to achieve breaks. 3) Persistent breaks canbe a source of translocations. Coupling endonucleases to end-processingenzymes prevents the establishment of a persistent break and reduces theincidence of gross chromosomal rearrangements, thereby potentiallyimproving the safety of endonuclease-induced targeted disruption. 4)Multiple changes in a single round of mutagenesis may be achieved, foruse for example, in multi-allelic knockouts and multiplexing, as datadescribed herein suggests that coupling endonucleases to end-processingenzymes improves the mutagenic rate of two given endonucleases 5-fold attheir respective targets, a 25-fold improvement may be realized indisrupting both targets simultaneously.

Any suitable method may be used to provide endonucleases, end-processingenzymes, and/or fusion proteins having endonuclease and end-processingactivity to host cells. In some embodiments one or more polypeptideshaving endonuclease and/or end-processing activity may be provideddirectly to cells. In some embodiments, expression of endonucleases,end-processing enzymes and/or fusion proteins having endonuclease andend-processing activity in a host cell can result from delivery of oneor more polynucleotides encoding one or more endonucleases,end-processing enzymes, and/or fusion proteins having endonuclease andend-processing activity to the host cell. In some embodiments, one ormore polynucleotides is a DNA expression vector. In some embodiments,one or more polynucleotides is an RNA expression vector. In someembodiments, trans-splicing, polypeptide cleavage and/or polypeptideligation can be involved in expression of one or more proteins in acell. Methods for polynucleotide and polypeptide delivery to cells arewell known in the art.

The compositions and methods described herein are useful for generatingtargeted disruptions of the coding sequences of genes and in someembodiments, creating gene knockouts. Targeted cleavage by thecompositions and methods described herein can also be used to alternon-coding sequences (e.g., regulatory sequences such as promoters,enhancers, initiators, terminators, splice sites) to alter the levels ofexpression of a gene product. Such methods can be used, for example, forbiological research, for biotechnology applications such as cropmodification, for therapeutic purposes, functional genomics, and/ortarget validation studies.

Targeted mutations resulting from the methods and compositions describedherein include, but are not limited to, point mutations (e.g.,conversion of a single base pair to a different base pair),substitutions (e.g., conversion of a plurality of base pairs to adifferent sequence of identical length), insertions of one or more basepairs, deletions of one or more base pairs and any combination of theaforementioned sequence alterations.

Some embodiments relate to coupling the activity of one or moresite-specific endonucleases with one or more end-processing enzymes. Insome embodiments, the endonucleases and end-processing enzymes areprovided as separate proteins. In some embodiments, the endonucleasesand end-processing enzymes are co-expressed in a cell. If expression ofthe separate endonucleases and end-processing enzymes is bypolynucleotide delivery, each of the endonucleases and end-processingenzymes can be encoded by separate polynucleotides, or by a singlepolynucleotide. In some embodiments, the endonucleases andend-processing enzymes are encoded by a single polynucleotide andexpressed by a single promoter. In some embodiments, an endonuclease andend-processing enzymes are linked by a T2A sequence which allows for twoseparate proteins to be produced from a single translation. In someembodiments, a different linker sequence can be used. In otherembodiments a single polynucleotide encodes the endonucleases andend-processing enzymes separated by an Internal Ribosome Entry Sequence(IRES).

Several embodiments relate to coupling the activity of one or moresite-specific endonucleases selected from the group consisting of:homing endonucleases (meganucleases) (including engineered homingedonucleases), zinc finger nucleases, and TAL effector nucleases withone or more end-processing enzymes. The endonucleases may compriseheterologous DNA-binding and cleavage domains (e.g., zinc fingernucleases; homing endonuclease DNA-binding domains with heterologouscleavage domains or TAL-effector domain nuclease fusions) or,alternatively, the DNA-binding domain of a naturally-occurring nucleasemay be altered to bind to a selected target site (e.g., a homingendonuclease that has been engineered to bind to site different than thecognate binding site or a TAL-effector domain nuclease fusion). In someembodiments, the endonucleases and end-processing enzymes are providedas a fusion protein. In some embodiments, the endonucleases andend-processing enzymes are provided as separate proteins. In someembodiments, the endonucleases and end-processing enzymes areco-expressed in a cell.

Several embodiments relate to coupling the activity of one or moresite-specific homing endonucleases selected from the group consistingof: I-AniI, I-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI,I-PanII, I-PanMI, I-SceII, I-PpoI, I-SceIII, I-CreI, I-LtrI, I-GpiI,I-GZeI, I-OnuI, I-HjeMI, I-TevI, I-TevII, and I-TevIII with one or moreDNA end-processing enzymes selected from the group consisting of: Trex2,Trex1, Trex1 without transmembrane domain, Apollo, Artemis, DNA2, Exo1,ExoT, ExoIII, Fen1, Fan1, MreII, Rad2, Rad9, TdT (terminaldeoxynucleotidyl transferase), PNKP, RecE, RecJ, RecQ, Lambdaexonuclease, Sox, Vaccinia DNA polymerase, exonuclease I, exonucleaseIII, exonuclease VII, NDK1, NDK5, NDK7, NDK8, WRN, T7-exonuclease Gene6, avian myeloblastosis virus integration protein (IN), Bloom, AntarticPhophatase, Alkaline Phosphatase, Poly nucleotide Kinase (PNK), ApeI,Mung Bean nuclease, Hex1, TTRAP (TDP2), Sgs1, Sae2, CtIP, Pol mu, Pollambda, MUS81, EME1, EME2, SLX1, SLX4 and UL-12. In some embodiments,the homing endonucleases and DNA end-processing enzymes are provided asa fusion protein. In some embodiments, the endonucleases and DNAend-processing enzymes are provided as separate proteins. In someembodiments, the endonucleases and DNA end-processing enzymes areco-expressed in a host cell.

Several embodiments relate to coupling the activity of one or more ZFNswith one or more DNA end-processing enzymes selected from the groupconsisting of: Trex2, Trex1, Trex1 without transmembrane domain, Apollo,Artemis, DNA2, Exo1, ExoT, ExoIII, Fen1, Fan1, MreII, Rad2, Rad9, TdT(terminal deoxynucleotidyl transferase), PNKP, RecE, RecJ, RecQ, Lambdaexonuclease, Sox, Vaccinia DNA polymerase, exonuclease I, exonucleaseIII, exonuclease VII, NDK1, NDK5, NDK7, NDK8, WRN, T7-exonuclease Gene6, avian myeloblastosis virus integration protein (IN), Bloom, AntarticPhophatase, Alkaline Phosphatase, Poly nucleotide Kinase (PNK), ApeI,Mung Bean nuclease, Hex1, TTRAP (TDP2), Sgs1, Sae2, CtIP, Pol mu, Pollambda, MUS81, EME1, EME2, SLX1, SLX4 and UL-12. In some embodiments,the ZFNs and DNA end-processing enzymes are provided as a fusionprotein. In some embodiments, the ZFNs and DNA end-processing enzymesare provided as separate proteins. In some embodiments, the ZFNs and DNAend-processing enzymes are co-expressed in a host cell.

Several embodiments relate to coupling the activity of one or moreTALENs with one or more DNA end-processing enzymes selected from thegroup consisting of: Trex2, Trex1, Trex1 without transmembrane domain,Apollo, Artemis, DNA2, Exo1, ExoT, ExoIII, Fen1, Fan1, MreII, Rad2,Rad9, TdT (terminal deoxynucleotidyl transferase), PNKP, RecE, RecJ,RecQ, Lambda exonuclease, Sox, Vaccinia DNA polymerase, exonuclease I,exonuclease III, exonuclease VII, NDK1, NDK5, NDK7, NDK8, WRN,T7-exonuclease Gene 6, avian myeloblastosis virus integration protein(IN), Bloom, Antartic Phophatase, Alkaline Phosphatase, Poly nucleotideKinase (PNK), ApeI, Mung Bean nuclease, Hex1, TTRAP (TDP2), Sgs1, Sae2,CtIP, Pol mu, Pol lambda, MUS81, EME1, EME2, SLX1, SLX4 and UL-12. Insome embodiments, the TALENs and DNA end-processing enzymes are providedas a fusion protein. In some embodiments, the TALENs and DNAend-processing enzymes are provided as separate proteins. In someembodiments, the TALENs and DNA end-processing enzymes are co-expressedin a host cell.

In several embodiments, the activity of one or more site-specific homingendonucleases selected from the group consisting of: I-AniI, I-SceI,I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-PanII, I-PanMI,I-SceII, I-PpoI, I-SceIII, I-CreI, I-LtrI, I-GpiI, I-GZeI, I-OnuI,I-HjeMI, I-TevI, I-TevII, and I-TevIII is coupled with the activity ofone or more DNA end-processing enzymes selected from the groupconsisting of: Apollo, Artemis, Dna2, Exo1, Mre11, Rad2, RecE, Lambdaexonuclease, Sox, exonuclease VII, T7-exonuclease Gene 6 and UL-12. Insome embodiments, the homing endonucleases and DNA end-processingenzymes are provided as a fusion protein. In some embodiments, theendonucleases and DNA end-processing enzymes are provided as separateproteins. In some embodiments, the endonucleases and DNA end-processingenzymes are co-expressed in a host cell.

In several embodiments, the activity of one or more site-specific homingendonucleases selected from the group consisting of: I-AniI, I-SceI,I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-PanII, I-PanMI,I-SceII, I-PpoI, I-SceIII, I-CreI, I-LtrI, I-GpiI, I-GZeI, I-OnuI,I-HjeMI, I-TevI, I-TevII, and I-TevIII is coupled with the activity ofone or more DNA end-processing enzymes selected from the groupconsisting of: Sox and UL-12. In some embodiments, the homingendonucleases and DNA end-processing enzymes are provided as a fusionprotein. In some embodiments, the endonucleases and DNA end-processingenzymes are provided as separate proteins. In some embodiments, theendonucleases and DNA end-processing enzymes are co-expressed in a hostcell.

In several embodiments, the activity of one or more site-specific homingendonucleases selected from the group consisting of: I-AniI, I-SceI,I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-PanII, I-PanMI,I-SceII, I-PpoI, I-SceIII, I-CreI, I-LtrI, I-GpiI, I-GZeI, I-OnuI,I-HjeMI, I-TevI, I-TevII, and I-TevIII is coupled with the activity ofone or more DNA end-processing enzymes selected from the groupconsisting of: Trex2, Vaccinia DNA polymerase, Mre11, exonuclease I,exonuclease III, NDK1, NDK5, NDK7, NDK8, and WRN. In some embodiments,the homing endonucleases and DNA end-processing enzymes are provided asa fusion protein. In some embodiments, the endonucleases and DNAend-processing enzymes are provided as separate proteins. In someembodiments, the endonucleases and DNA end-processing enzymes areco-expressed in a host cell.

In several embodiments, the activity of one or more site-specific homingendonucleases selected from the group consisting of: I-AniI, I-SceI,I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-PanII, I-PanMI,I-SceII, I-PpoI, I-SceIII, I-CreI, I-LtrI, I-GpiI, I-GZeI, I-OnuI,I-HjeMI, I-TevI, I-TevII, and I-TevIII is coupled with the activity ofFen1. In some embodiments, the homing endonucleases and DNAend-processing enzymes are provided as a fusion protein. In someembodiments, the endonucleases and DNA end-processing enzymes areprovided as separate proteins. In some embodiments, the endonucleasesand DNA end-processing enzymes are co-expressed in a host cell.

In several embodiments, the activity of one or more site-specific homingendonucleases selected from the group consisting of: I-AniI, I-SceI,I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-PanII, I-PanMI,I-SceII, I-PpoI, I-SceIII, I-CreI, I-LtrI, I-GpiI, I-GZeI, I-OnuI,I-HjeMI, I-TevI, I-TevII, and I-TevIII is coupled with the activity ofTdT. In some embodiments, the homing endonucleases and DNAend-processing enzymes are provided as a fusion protein. In someembodiments, the endonucleases and DNA end-processing enzymes areprovided as separate proteins. In some embodiments, the endonucleasesand DNA end-processing enzymes are co-expressed in a host cell.

Some embodiments relate to coupling the activity of multiplesite-specific endonucleases with the activity of one or moreend-processing enzymes. The site specific endonucleases may cleavetarget sites within the same gene or in different genes. In someembodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 site-specificendonucleases may be provided to a cell along with one or moreend-processing enzymes. In some embodiments, a combination of homingendonucleases, zinc finger endonucleases, and/or TAL effectorendonucleases may be provided to a cell with one or more end-processingenzymes. In some embodiments, the end-processing enzyme is anexonuclease. In some embodiments, a 5′ and a 3′ exonuclease may beprovided. If expression of the multiple endonucleases and one or moreexonucleases is by polynucleotide delivery, each of the endonucleasesand exonucleases can be encoded by separate polynucleotides, or by asingle polynucleotide. In some embodiments, the endonucleases andexonucleases are encoded by a single polynucleotide and expressed by asingle promoter. In some embodiments, the endonucleases and exonucleasesare linked by a T2A sequence which allows for separate proteins to beproduced from a single translation. In some embodiments, differentlinker sequences can be used. In other embodiments, a singlepolynucleotide encodes the endonucleases and exonucleases separated byIRESs.

Several embodiments relate to a heterologous fusion protein, whichcomprises an endonuclease domain and an end-processing domain orportions thereof. Several embodiments relate to a heterologous fusionconstruct, which encodes a fusion protein having endonuclease andend-processing activity. The present embodiments also relate to vectorsand host cells comprising the heterologous fusion construct as well asmethods for producing a fusion protein having endonuclease andend-processing activity and compositions thereof. In one embodiment, theendonuclease domain is coupled to the end-processing domain byrecombinant means (e.g., the fusion protein is generated by translationof a nucleic acid in which a polynucleotide encoding all or a portion ofa endonuclease is joined in-frame with a polynucleotide encoding all ora portion of a end-processing enzyme). In other embodiments, theendonuclease domain and end-processing domain of a fusion protein may belinked chemically. This chemical linkage can be carried out, forexample, by using bifunctional linker molecules, such as, BS3(Bis[sulfosuccinimidyl] suberate).

Some embodiments relate to a fusion protein comprising an endonucleasedomain and exonuclease domain. In some embodiments the fusion proteincomprises at least a fragment or variant of a homing endonuclease and atleast a fragment or variant of an exonuclease, for example a 3′exonuclease, which are associated with one another by genetic orchemical conjugation to one another. In several embodiments, the 3′exonuclease is a Trex2 monomer, dimer, or a variant thereof. In otherembodiments, the fusion protein comprises at least a fragment or variantof a zinc finger endonuclease and at least a fragment or variant of a 5′exonuclease, which are associated with one another, by genetic fusion orchemical conjugation to one another. The endonuclease and exonuclease,once part of the fusion protein, may be referred to as a “portion”,“region,” “domain” or “moiety” of the endo/exo-nuclease fusion protein.

In some embodiments, an end-processing enzyme (or fragment or variantthereof) is fused directly to an endonuclease (or fragment or variantthereof). The end-processing enzyme (or fragment or variant thereof) maybe fused to the amino terminus or the carboxyl terminus of theendonuclease (or fragment or variant thereof).

An endonuclease/end-processing enzyme fusion protein may optionallyinclude a linker peptide between the endonuclease and end-processingenzyme domains to provide greater physical separation between themoieties and thus maximize the accessibility of the endonucleaseportion, for instance, for binding to its target sequence. The linkerpeptide may consist of amino acids selected to make it more flexible ormore rigid depending on the relevant function. The linker sequence maybe cleavable by a protease or cleavable chemically to yield separateendonuclease and end-processing enzyme moieties. Examples of enzymaticcleavage sites in the linker include sites for cleavage by a proteolyticenzyme, such as enterokinase, Factor Xa, trypsin, collagenase, andthrombin. In some embodiments, the protease is one which is producednaturally by the host or it is exogenously introduced. Alternatively,the cleavage site in the linker may be a site capable of being cleavedupon exposure to a selected chemical, e.g., cyanogen bromide,hydroxylamine, or low pH. The optional linker sequence may serve apurpose other than the provision of a cleavage site. The linker sequenceshould allow effective positioning of the endonuclease moiety withrespect to the end-processing enzyme moiety so that the endonucleasedomain can recognize and cleave its target sequence and theend-processing domain can modify the DNA ends exposed at the cleavagesite. The linker may also be a simple amino acid sequence of asufficient length to prevent any steric hindrance between theendonuclease domain and the end-processing domain. In addition, thelinker sequence may provide for post-translational modificationincluding, but not limited to, e.g., phosphorylation sites,biotinylation sites, sulfation sites, γ-carboxylation sites, and thelike.

In some embodiments the linker sequence comprises from about 4 to 30amino acids, more preferably from about 8 to 22 amino acids. That is,the linker sequence can be any number of amino acids from about 4 to 30,such as at least or equal to 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 aminoacids. In some embodiments, the linker sequence is flexible so as nothold the biologically active peptide in a single undesired conformation.The linker may be predominantly comprised of amino acids with small sidechains, such as glycine, alanine, and serine, to provide forflexibility. In some embodiments about 80 or 90 percent or greater ofthe linker sequence comprises glycine, alanine, or serine residues,particularly glycine and serine residues. In several embodiments, a G4Slinker peptide separates the end-processing and endonuclease domains ofthe fusion protein. In other embodiments, a T2A linker sequence allowsfor two separate proteins to be produced from a single translation.Suitable linker sequences can be readily identified empirically.Additionally, suitable size and sequences of linker sequences also canbe determined by conventional computer modeling techniques.

Expression of a fusion protein in a cell can result from delivery of thefusion protein to the cell or by delivery of a polynucleotide encodingthe fusion protein to a cell, wherein the polynucleotide is transcribed,and the transcript is translated, to generate the fusion protein.Trans-splicing, polypeptide cleavage and polypeptide ligation can alsobe involved in expression of a protein in a cell. Methods forpolynucleotide and polypeptide delivery to cells are well known in theart.

A variety of DNA molecules encoding the above-described endonucleases,end-processing enzymes and fusion proteins may be constructed forproviding the selected proteins or peptides to a cell. The DNA moleculesencoding the endonucleases, end-processing enzyme, and fusion proteinsmay be modified to contain different codons to optimize expression in aselected host cell, as is known in the art.

A variety of RNA molecules encoding the above-described endonucleases,end-processing enzymes and fusion proteins may be constructed forproviding the selected proteins or peptides to a cell. The RNA moleculesencoding the endonucleases, end-processing enzyme, and fusion proteinsmay be modified to contain different codons to optimize expression in aselected host cell, as is known in the art.

Several embodiments relate to the prevention of precise cNHEJ mediatedrepair of endonuclease-induced double strand breaks by simultaneousexpression of end-processing enzymes capable of recognizing thepost-endonuclease break structure, resulting in the modification of DNAends prior to ligation, promoting a mutagenic outcome. Some embodimentsrelate to the simultaneous expression exonucleases capable ofrecognizing the post-endonuclease break structure, resulting in thetrimming of DNA ends prior to ligation, promoting a mutagenic outcome.Simultaneous expression of a site-specific endonuclease and anend-processing enzyme improves the efficiency of targeted genedisruption by up to ˜70 fold, essentially fixing a mutagenic outcome in100% of a population of cells containing the target site in less than 72hours.

In some embodiments, effective amounts of endonucleases andend-processing enzymes or an effective amount of a fusion protein aredelivered to a cell either directly by contacting the cell will theprotein(s) or by transient expression from an expression construct. Insuch embodiments, cell division reduces the concentration of thenucleases to sub-active levels within a few cell divisions.

Several embodiments relate to a method of conferring site specificity ona DNA end-processing enzyme by physically tethering an end-processingenzyme domain to a site specific DNA binding domain. In someembodiments, the end-processing enzyme domain is tethered to a DNAbinding domain through a linker peptide. The composition and structureof the linker peptide is not especially limited and in some embodimentsthe linker may be chemically or enzymaticly cleavable. The linkerpeptide may be flexible or rigid and may comprise from about 4 to 30amino acids. In other embodiments, the end-processing enzyme domain ischemically fused to a DNA binding domain. Not wishing to be bound by aparticular theory, imparting site specificity to a end-processing enzymethrough tethering the end-processing enzyme to a site specific DNAbinding domain decreases toxicity associated with indiscriminateend-processing activity, such as exonuclease activity, and reduces theeffective amount of end-processing enzyme required for efficientmodification of the exposed double stranded DNA break caused byendonuclease activity compared to untethered end-processing enzyme. Insome embodiments, the end-processing enzyme is tethered to a homingendonuclease. In other embodiments, the end-processing enzyme istethered to zinc finger endonuclease. In some embodiments, anend-processing enzyme domain is tethered to a zinc finger DNA bindingdomain which binds to a DNA sequence adjacent to the cleavage site of ahoming endonuclease or zinc finger endonuclease.

Several embodiments relate to coupling the activity of one or moresite-specific endonucleases with Trex2. Trex2 may be provided as amonomer or dimer. The Trex2 enzyme specifically hydrolyzes thephosphodiester bonds which are exposed at 3′ overhangs. While the homingendonucleases generate 3′ overhangs which are susceptible to Trex2exonuclease activity, the zinc finger nucleases, which utilize the Fok1cleavage domain, generate double strand DNA breaks with 5′ overhangs.The homing endonucleases and zinc finger nucleases generate mutations attheir cleavage sites at a baseline rate. Co-expression of Trex2 withhoming endonucleases increased the mutation rate ˜70 fold. Co-expressionof Trex2 with zinc finger endonucleases was also observed to effect onthe rate of mutation. See FIGS. 11A and 11B. Accordingly, severalembodiments described herein relate to improving the mutation rateassociated zinc finger endonuclease targeted cleavage events by couplingzinc finger endonuclease to exonucleases which cleave 5′ overhangs. Someembodiments relate to coupling 3′ exonucleases to zinc fingerendonucleases wherein the nuclease domain of the zinc fingerendonuclease generates 3′ overhangs.

Some embodiments relate to the co-expression of a homing endonucleaseand the exonuclease, Trex2, via a single promoter linked by a T2Asequence that enables separate polypeptides to be produced from a singletranslation event. In this way, the endonuclease and exonuclease areprovided in a 1 to 1 ratio. Higher rates of modification are achievedusing T2A linked expression of the homing endonuclease, I-SceI, andTrex2 than is achieved through co-transduction of separate I-SceI, andTrex2 expression constructs. In some embodiments, a fusion proteincomprising one or more endonuclease domains and one or more Trex2domains may be provided.

In another aspect, methods of co-expressing an end-processing enzymewith a zinc finger endonuclease capable of mutating the CCR-5 geneand/or inactivating CCR-5 function in a cell or cell line are provided.In some embodiments, a method for improving the inactivation of a CCR-5gene in a human cell is provided, the method comprising administering tothe cell any site specific endonuclease having a target site in a CCR5coupled to an end-processing enzyme. In some embodiments, a method forimproving the inactivation of a CCR-5 gene in a human cell is provided,the method comprising administering to the cell any site specificendonuclease having a target site in a CCR5 coupled to an exonucleasecapable of cleaving the phosphodiester bonds created at the site ofendonuclease cleavage. In some embodiments, a method for improving theinactivation of a CCR-5 gene in a human cell is provided, the methodcomprising administering to the cell any site specific endonucleasehaving a target site in a CCR5 and contemporaneously administering anexonuclease capable of cleaving the phosphodiester bonds created at thesite of endonuclease cleavage. Examples of suitable endonucleasesinclude engineered homing endonucleases and meganucleases, which havevery long recognition sequences, some of which are likely to be present,on a statistical basis, once in a human-sized genome. Any such nucleasehaving a unique target site in a CCR5 gene can be used instead of, or inaddition to, a zinc finger nuclease, in conjunction with an exonucleasefor targeted cleavage in a CCR5 gene. Some embodiments relate toadministration of a fusion protein comprising a CCR5-site-specificendonuclease and an exonuclease capable of cleaving the phosphodiesterbonds created at the site of endonuclease cleavage.

Expression Vectors

Expression constructs can be readily designed using methods known in theart. Examples of nucleic acid expression vectors include, but are notlimited to: recombinant viruses, lentiviruses, adenoviruses, plasmids,bacterial artificial chromosomes, yeast artificial chromosomes, humanartificial chromosomes, minicircle DNA, episomes, cDNA, RNA, and PCRproducts. In some embodiments, nucleic acid expression vectors encode asingle peptide (e.g., an endonuclease, an end-processing enzyme, or afusion protein having endonuclease and end-processing activity). In someembodiments, nucleic acid expression vectors encode one or moreendonucleases and one or more end-processing enzymes in a single,polycistronic expression cassette. In some embodiments, one or moreendonucleases and one or more end-processing enzymes are linked to eachother by a 2A peptide sequence or equivalent “autocleavage” sequence. Insome embodiments, a polycistronic expression cassette may incorporateone or more internal ribosomal entry site (IRES) sequences between openreading frames. In some embodiments, the nucleic acid expression vectorsare DNA expression vectors. In some embodiments, the nucleic acidexpression vectors are RNA expression vectors.

In some embodiments, a nucleic acid expression vector may furthercomprise one or more selection markers that facilitate identification orselection of host cells that have received and express theendonuclease(s), end-processing enzyme(s), and/or fusion protein(s)having endonuclease and end-processing activity along with the selectionmarker. Examples of selection markers include, but are not limited to,genes encoding fluorescent proteins, e.g., EGFP, DS-Red, YFP, and CFP;genes encoding proteins conferring resistance to a selection agent,e.g., Puro^(R) gene, Zeo^(R) gene, Hygro^(R) gene, neo^(R) gene, and theblasticidin resistance gene. In some cases, the selection markercomprises a fluorescent reporter and a selection marker.

In some embodiments, a DNA expression vector may comprise a promotercapable of driving expression of one or more endonuclease(s),end-processing enzyme(s), and/or fusion protein(s) having endonucleaseand end-processing activity. Examples of promoters include, but are notlimited to, retroviral LTR elements; constitutive promoters such as CMV,HSV1-TK, SV40, EF-1α, β-actin; inducible promoters, such as thosecontaining Tet-operator elements; and tissue specific promoters.Suitable bacterial and eukaryotic promoters are well known in the artand described, e.g., in Sambrook et al., Molecular Cloning, A LaboratoryManual (2nd ed. 2001); Kriegler, Gene Transfer and Expression: ALaboratory Manual (1990); and Current Protocols in Molecular Biology(2010). Non-limiting examples of plant promoters include promotersequences derived from A. thaliana ubiquitin-3 (ubi-3).

In some embodiments, a nucleic acid encoding one or more endonucleases,end-processing enzymes, and/or fusion proteins having endonuclease andend-processing activity can be cloned into a vector for transformationinto prokaryotic or eukaryotic cells. In some embodiments, nucleic acidsencoding different endonucleases and end-processing enzymes are clonedinto the same vector. In such cases, the nucleic acids encodingdifferent endonucleases and end-processing enzymes may optionally beseparated by T2A or IRES sequences. Vectors can be prokaryotic vectors,e.g., plasmids, or shuttle vectors, insect vectors, or eukaryoticvectors, including plant vectors described herein. Expression of thenucleases and fusion proteins may be under the control of a constitutivepromoter or an inducible promoter.

Introduction of polypeptides having endonuclease and/or end-processingactivity and/or polynucleotides encoding polypeptides havingendonuclease and/or end-processing activity into host cells may use anysuitable methods for nucleic acid or protein delivery as describedherein or as would be known to one of ordinary skill in the art. Thepolypeptides and polynucleotides described herein can be delivered intocultured cells in vitro, as well as in situ into tissues and wholeorganisms. Introduction of the polypeptides and polynucleotides of thepresent embodiments into a host cell can be accomplished chemically,biologically, or mechanically. This may include, but is not limited to,electroporation, sonoporation, use of a gene gun, lipotransfection,calcium phosphate transfection, use of dendrimers, microinjection,polybrene, protoplast fusion, the use of viral vectors includingadenoviral, AAV, and retroviral vectors, and group II ribozymes.

Organisms

The present invention is applicable to any prokaryotic or eukaryoticorganism in which it is desired to create a targeted genetic mutation.Examples of eukaryotic organisms include, but are not limited to, algae,plants, animals (e.g., mammals such as mice, rats, primates, pigs, cows,sheep, rabbits, etc.), fish, and insects. In some embodiments, isolatedcells from the organism can be genetically modified as described herein.In some embodiments, the modified cells can develop into reproductivelymature organisms. Eukaryotic (e.g., algae, yeast, plant, fungal,piscine, avian, and mammalian cells) cells can be used. Cells fromorganisms containing one or more additional genetic modifications canalso be used.

Examples of mammalian cells include any cell or cell line of theorganism of interest, for example oocytes, somatic cells, K562 cells,CHO (Chinese hamster ovary) cells, HEP-G2 cells, BaF-3 cells, Schneidercells, COS cells (monkey kidney cells expressing SV40 T-antigen), CV-1cells, HuTu80 cells, NTERA2 cells, NB4 cells, HL-60 cells and HeLacells, 293 cells and myeloma cells like SP2 or NS0. Peripheral bloodmononucleocytes (PBMCs) or T-cells can also be used, as can embryonicand adult stem cells. For example, stem cells that can be used includeembryonic stem cells (ES), induced pluripotent stem cells (iPSC),mesenchymal stem cells, hematopoietic stem cells, muscle stem cells,skin stem cells, and neuronal stem cells.

Examples of target plants and plant cells include, but are not limitedto, monocotyledonous and dicotyledonous plants, such as crops includinggrain crops (e.g., wheat, maize, rice, millet, barley), fruit crops(e.g., tomato, apple, pear, strawberry, orange), forage crops (e.g.,alfalfa), root vegetable crops (e.g., carrot, potato, sugar beets, yam),leafy vegetable crops (e.g., lettuce, spinach); flowering plants (e.g.,petunia, rose, chrysanthemum), conifers and pine trees (e.g., pine fir,spruce); plants used in phytoremediation (e.g., heavy metal accumulatingplants); oil crops (e.g., sunflower, rape seed) and plants used forexperimental purposes (e.g., Arabidopsis). Thus, the disclosed methodsand compositions have use over a broad range of plants, including, butnot limited to, species from the genera Asparagus, Avena, Brassica,Citrus, Citrullus, Capsicum, Cucurbita, Daucus, Erigeron, Glycine,Gossypium, Hordeum, Lactuca, Lolium, Lycopersicon, Malus, Manihot,Nicotiana, Orychophragmus, Oryza, Persea, Phaseolus, Pisum, Pyrus,Prunus, Raphanus, Secale, Solanum, Sorghum, Triticum, Vitis, Vigna, andZea. The term plant cells include isolated plant cells as well as wholeplants or portions of whole plants such as seeds, callus, leaves, roots,etc. The present disclosure also encompasses seeds of the plantsdescribed above. The present disclosure further encompasses the progeny,clones, cell lines, or cells of the plants described.

Generating Homozygously Modified Organisms

Cells in which one or more endonucleases are co-expressed with one ormore end-processing enzyme(s) and/or cells in which one or more fusionproteins having endonuclease and end-processing activity are expressedare then assayed for the presence of mutations at the endonucleasecleavage site(s). Such modified cells can be identified using anysuitable method known to the skilled artisan, including sequencing, PCRanalysis, southern blotting, and the like. In some embodiments, anamplicon spanning the endonuclease target site is generated by PCR. Theamplicon is then exposed to the endonuclease and the ability of theendonuclease to cut the amplicon is assessed. Mutation of the targetsite is indicated by the absence of endonuclease generated cleavageproducts.

Subsequently, cells containing the mutated target site(s) are culturedor otherwise treated such that they generate a whole organism with themutated target site. For example, traditional methods of pro-nuclearinjection or oocyte injection can be used to generate animals with themutated target site. Likewise, plant cells containing the mutated targetsite(s) can be cultured to regenerate a whole plant which possesses themutant genotype and thus the desired phenotype. Regeneration can also beobtained from plant callus, explants, organs, pollens, embryos, or partsthereof. Once the heterozygous organisms containing the mutated targetsite(s) reach reproductive maturity, they can be crossed to each other,or in some instances, spores may be grown into haploids. Of theresulting progeny from crosses, approximately 25% will be homozygousmutant/mutant at the target locus.

Pharmaceutical Compositions and Administration

Endonucleases, end-processing enzymes and fusion proteins havingendonuclease and end-processing activity and expression vectors encodingendonucleases, end-processing enzymes and fusion proteins havingendonuclease and end-processing activity can be administered directly toa patient for targeted cleavage of a DNA sequence and for therapeutic orprophylactic applications, for example, cancer, ischemia, diabeticretinopathy, macular degeneration, rheumatoid arthritis, psoriasis, HIVinfection, sickle cell anemia, Alzheimer's disease, muscular dystrophy,neurodegenerative diseases, vascular disease, cystic fibrosis, stroke,hyper IGE syndrome, hemophilia and the like. In some embodiments, thecompositions described herein (e.g., endonucleases, end-processingenzymes and fusion proteins having endonuclease and end-processingactivity and expression vectors encoding endonucleases, end-processingenzymes and fusion proteins having endonuclease and end-processingactivity) can be used in methods of treating, preventing, or inhibitinga disease (e.g., cancer, ischemia, diabetic retinopathy, maculardegeneration, rheumatoid arthritis, psoriasis, HIV infection, sicklecell anemia, Alzheimer's disease, muscular dystrophy, neurodegenerativediseases, vascular disease, cystic fibrosis, stroke, hyper IGE syndrome,hemophilia) or ameliorating a disease condition or symptom associatedwith a disease, such as, cancer, ischemia, diabetic retinopathy, maculardegeneration, rheumatoid arthritis, psoriasis, HIV infection, sicklecell anemia, Alzheimer's disease, muscular dystrophy, neurodegenerativediseases, vascular disease, cystic fibrosis, stroke, hyper IGE syndrome,hemophilia. In some embodiments endonucleases, end-processing enzymesand fusion proteins having endonuclease and end-processing activity andexpression vectors encoding endonucleases, end-processing enzymes andfusion proteins having endonuclease and end-processing activity areadministered to treat, prevent, or inhibit an autosomal dominantdisease, such as achondroplasia, pseudoachondroplasia, the multipleepiphyseal dysplasias, chondrodysplasias, osteogenesis imperfecta,Marfan syndrome, polydactyly, hereditary motor sensory neuropathies Iand II (Charcot-Marie-Tooth disease), myotonic dystrophy, andneurofibromatosis or ameliorate a disease condition or symptomassociated with an autosomal dominant disease, such as achondroplasia,pseudoachondroplasia, the multiple epiphyseal dysplasias,chondrodysplasias, osteogenesis imperfecta, Marfan syndrome,polydactyly, hereditary motor sensory neuropathies I and II(Charcot-Marie-Tooth disease), myotonic dystrophy, andneurofibromatosis. In some embodiments endonucleases, end-processingenzymes and fusion proteins having endonuclease and end-processingactivity and expression vectors encoding endonucleases, end-processingenzymes and fusion proteins having endonuclease and end-processingactivity are administered to treat, prevent, or inhibit a disease causedby misregulation of genes. In some embodiments endonucleases,end-processing enzymes and fusion proteins having endonuclease andend-processing activity and expression vectors encoding endonucleases,end-processing enzymes and fusion proteins having endonuclease andend-processing activity are administered to treat, prevent, or inhibit acancer, such as BCL-2, Bcl-XI, and FLIP, or ameliorate a diseasecondition or symptom associated with a cancer, such as BCL-2, Bcl-XI,and FLIP, by, for example, increasing the mutation rate of genes withanti-apoptotic activity.

Examples of microorganisms that can be inhibited (e.g., inhibiting thegrowth or infection) by provision of endonucleases, end-processingenzymes and fusion proteins having endonuclease and end-processingactivity include pathogenic bacteria, e.g., chlamydia, rickettsialbacteria, mycobacteria, staphylococci, streptococci, pneumococci,meningococci and conococci, klebsiella, proteus, serratia, pseudomonas,legionella, diphtheria, salmonella, bacilli, cholera, tetanus, botulism,anthrax, plague, leptospirosis, and Lyme disease bacteria; infectiousfungus, e.g., Aspergillus, Candida species; protozoa such as sporozoa(e.g., Plasmodia), rhizopods (e.g., Entamoeba) and flagellates(Trypanosoma, Leishmania, Trichomonas, Giardia, etc.); viral diseases,e.g., hepatitis (A, B, or C), herpes virus (e.g., VZV, HSV-1, HSV-6,HSV-II, CMV, and EBV), HIV, Ebola, adenovirus, influenza virus,flaviviruses, echovirus, rhinovirus, coxsackie virus, coronavirus,respiratory syncytial virus, mumps virus, rotavirus, measles virus,rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus,papillomavirus, poliovirus, rabies virus, and arboviral encephalitisvirus, etc.

Administration of therapeutically effective amounts is by any of theroutes normally used for introducing homing endonucleases or zinc fingerendonucleases into ultimate contact with the tissue to be treated. Theendonucleases, end-processing enzymes and fusion proteins havingendonuclease and end-processing activity are administered in anysuitable manner, and in some embodiments with pharmaceuticallyacceptable carriers. Suitable methods of administering such proteins orpolynucleotides are available and well known to those of skill in theart, and, although more than one route can be used to administer aparticular composition, a particular route can often provide a moreimmediate and more effective reaction than another route.

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositions that areavailable (see, e.g., Remington's Pharmaceutical Sciences).

The endonucleases, end-processing enzymes and fusion proteins havingendonuclease and end-processing activity or vectors encodingendonucleases, end-processing enzymes and fusion proteins havingendonuclease and end-processing activity, alone or in combination withother suitable components, can be made into aerosol formulations (e.g.,they can be “nebulized”) to be administered via inhalation. Aerosolformulations can be placed into pressurized acceptable propellants, suchas dichlorodifluoromethane, propane, nitrogen, and the like.

Formulations suitable for parenteral administration, such as, forexample, by intravenous, intramuscular, intradermal, and subcutaneousroutes, include aqueous and non-aqueous, isotonic sterile injectionsolutions, which can contain antioxidants, buffers, bacteriostats, andsolutes that render the formulation isotonic with the blood of theintended recipient, and aqueous and non-aqueous sterile suspensions thatcan include suspending agents, solubilizers, thickening agents,stabilizers, and preservatives. The disclosed compositions can beadministered, for example, by intravenous infusion, orally, topically,intraperitoneally, intravesically or intrathecally. The formulations ofcompounds can be presented in unit-dose or multi-dose sealed containers,such as ampules and vials. Injection solutions and suspensions can beprepared from sterile powders, granules, and tablets of the kindpreviously described.

Kits

Also provided are kits for performing any of the above methods. The kitstypically contain one or more endonucleases, end-processing enzymesand/or fusion proteins having endonuclease and end-processing activityor expression vectors encoding endonucleases, end-processing enzymesand/or fusion proteins having endonuclease and end-processing activityas described herein. The kits may also contain a reporter construct,such as the mCherry+ reporter construct described herein, containing acloning site for insertion of the target site for a selectedendonuclease of interest. In some embodiments, kits may contain one ormore plasmids according to SEQ ID NOs: 110-145. For example, kits forscreening mutagenesis produced by coupled endonuclease andend-processing activity and/or fusion proteins with activity to aparticular gene are provided with one or more reporter constructscontaining the desired target site(s). Similarly, kits for enrichingcells for a population of cells having a endonuclease-mediated genomicmodification may comprise a reporter construct comprising a target sitepresent in the genome of the cells and one or more endonuclease specificto the target site of interest and one or more selected end-processingenzymes and/or one or more fusion proteins specific to the target siteof interest.

The kits can also contain cells, buffers for transformation of cells,culture media for cells, and/or buffers for performing assays.Typically, the kits also contain a label, which includes any materialsuch as instructions, packaging or advertising leaflet that is attachedto or otherwise accompanies the other components of the kit.

While the foregoing written description enables one of ordinary skill tomake and use what is considered presently to be the best mode thereof,those of ordinary skill will understand and appreciate the existence ofvariations, combinations, and equivalents of the specific embodiment,method, and examples herein. The present embodiments should thereforenot be limited by the above described embodiment, method, and examples,but by all embodiments and methods within the scope and spirit of thepresent embodiments.

The following Examples are presented for the purposes of illustrationand should not be construed as limitations.

Example 1 Co-Expression of the Homing Endonuclease, I-SceI, and Trex2Exonuclease Increases the Rate at which I-SceI Induces Mutations

To determine if coupling an exonuclease with a site-specificendonuclease could enhance targeted gene disruption efficiency, weassessed the effect of Trex2 on the mutagenic repair of DSBs generatedby I-SceI. To ensure that Trex2 would be co-expressed with I-SceI, wedeveloped expression vectors that drive coupled expression of both anendonuclease and an end-processing enzyme from a single promoter via aT2A “skip” peptide motif. We also included mTagBFP fluorescent proteinco-expression by an internal ribosomal entry site (IRES) for trackingtransfection efficiency.

To measure the rate of nuclease-induced targeted disruption, a mutNHEJreporter construct (Traffic Light Reporter (TLR)) was constructed byplacing the I-SceI target site, SEQ ID NO: 1465′-AGTTACGCTAGGGATAACAGGGTAATATAG-3′, in front of the mCherryfluorescent protein ORF in the +3 reading frame. See FIG. 1A. When anendonuclease-induced DNA cleavage event results in a frameshift into the+3 reading frame, the mCherry fluorescent protein is placed in frame andcorrectly translated, resulting in red fluorescent cells that may beeasily detected by flow cytometry. HEK cell lines harboring the TLR weregenerated by plating 0.1×10⁶ HEK293 cells 24 hrs prior to transductionin a 24 well plate. mutNHEJ (TLR) reporter cell lines were made bytransducing HEK293 cells at limiting titer (˜5%) with ˜25 ngs of anintegrating lentivirus containing the reporter construct with 4 ug/mlpolybrene. Media was changed 24 hrs after transduction.

Expression vectors comprising the homing endonuclease, I-SceI, afluorescent protein (BFP), and optionally Trex2 with either a T2A or G4Slinker peptide were constructed according to the schematics provided inFIGS. 1B-H.

0.1×10̂6 HEK293 cells containing a genomically-integrated mutNHEJ (TLR)reporter cassette were plated 24 hrs prior to transfection in a 24 wellplate. The HEK 293 cells were transfected with expression constructscomprising the I-SceI mutant D44A alone, the I-SceI mutant D44A coupledto Trex2 via a T2A linker, I-SceI alone or I-SceI coupled to Trex2 via aT2A linker using Fugene transfection reagent according to manufacture'sprotocol. 72 hours following transduction of the cell line with theexpression vectors, the cells were analyzed by flow cytometry on a BDLSRII or BD FACS ARIAII. The mCherry fluorophore was excited using a 561nm laser and acquired with a 610/20 filter. The mTagBFP fluorophore wasexcited on a 405 nm laser with a 450/50 filter. Data was analyzed usingFlowJo software (FlowJo, Ashland Oreg.).

The plot shown in FIG. 2A demonstrates that I-SceI expression inducedmutagenic NHEJ events as visualized by mCherry+ expression and that therate of mutagenic NHEJ events (mCherry+) was significantly increasedfollowing co-expression of I-SceI with the exonuclease Trex2. See FIG.2A. While neither I-SceI D44A (catalytically inactive) nor I-SceI D44Acoupled to Trex2 was able to induce any measurable gene disruption,I-SceI coupled to Trex2 via T2A linkage exhibited a substantial increasein mCherry positive cells compared to I-SceI alone. See FIG. 2A.

Following co-expression of I-SceI endonuclease and Trex2 exonuclease,genomic DNA was extracted from the HEK 293 reporter cells using Qiagen'sDNA easy kit. Amplicons spanning the I-SceI target site were generatedby PCR, cloned into a shuttle vector and subjected to DNA sequencing ofthe I-SceI target site. The sequencing demonstrated that essentiallyevery cell in the population contains a mutated I-SceI target site, aspredicted by the reporter readout. See FIGS. 6A and 6B.

HEK 293 cells were transduced with expression constructs comprising theI-SceI mutant D44A alone, I-SceI alone or I-SceI coupled to Trex2 via aT2A linker. Following transduction of the cell line with the expressionvectors, the cells were analyzed by visual inspection daily. Live cellimages were taken 72 hours post transduction with the expressionvectors. The cells treated in each manner appeared indistinguishable,and there is no overt toxicity associated with Trex2 co-expression. SeeFIG. 21B.

To assess the total gene disruption rate, I-SceI and I-SceI-T2A-Trex2transfected cells were sorted based on varying BFP expression levels.HEK 293 cells containing a genomically-integrated cassette correspondingto the targeted disruption reporter illustrated in FIG. 1A (TLR) weretransduced with expression constructs comprising I-SceI-IRES-BFP (bluefluorescent protein) or I-SceI-T2A-Trex2-IRES-BFP. Expression ofI-SceI-IRES-BFP and I-SceI-T2A-Trex2-IRES-BFP was measured in thetransduced cells by a gating analysis of flow cytometry plots of BFPactivity. Cells with low, low-medium, medium and high levels of BFPexpression (corresponding to different levels of I-SceI endonuclease orI-SceI endonuclease/Trex2 exonuclease expression) were then assayed forinduced mutagenic NHEJ events as visualized by mCherry+ expression. Thedata demonstrated that low levels of I-SceI alone resulted in lowermutation levels, while expression of I-SceI in combination with Trex2result in high modification rates even at low levels of expression fromthe I-SceI-T2A-Trex2-IRES-BFP construct. See FIGS. 4A and 4B.

After the I-SceI and I-SceI-T2A-Trex2 transfected cells were sortedbased on varying BFP expression levels, the area flanking the I-SceItarget was amplified from each of the populations by PCR. 100 ng of eachPCR product was digested in vitro with recombinant I-SceI (New EnglandBiolabs) for 6 hours at 37° C. DNA was separated using a 1% agarose gelstained with ethidium bromide to look for a resistant band, indicativeof a mutagenic event at the locus that destroyed the I-SceI target site.See FIG. 5A. Percent disruption was calculated by quantifying bandintensity using Image J software, and dividing the intensity of theundigested band by the total. At low endonuclease expression levels, a25-fold increase in total gene disruption between I-SceI and I-SceIcoupled to Trex2 (2.2 to 50.2% respectively) was observed, and nearly100% of targets were disrupted in the medium and high expression gatesof I-SceI T2A Trex2 (90.3, and 97.1% respectively) See FIG. 5B.

These experiments indicate that while I-SceI exhibits a dose dependentincrease in gene disruption, I-SceI coupled to Trex2 quickly becomessaturated. Sequence analysis of the I-SceI target site in highexpressing cells confirmed that 100% of cells were modified in theI-SceI-T2A-Trex2 treated cells. See FIGS. 6A and 6B. Comparison of themutation spectra between I-SceI alone and I-SceI.T2A.Trex2 showed atrend towards small deletion events in the exonuclease treated cells.See FIGS. 6A, 6B and 7. In a kinetic analysis, while all constructsexhibited similar expression patterns, Trex2 expression coincided withthe appearance of disruption events at earlier time-points. See FIGS. 8Aand 8B. In sum, coupling of endonucleases to Trex2 expression in asingle open reading frame resulted in up to 25-fold enhancement in theefficiency of targeted gene disruption in cells from multiple speciesand in primary cell types, and is able to drive targeted knockout ratesto near completion within 72 hrs.

Example 2 Trex2 Exonuclease Increases the Mutation Rate of a Variety ofHoming Endonucleases

The applicability of Trex2-enhanced disruption to multiple differentnuclease scaffolds was evaluated. Targeted disruption reporter cassettes(mutNHEJ reporter cassettes) with target cleavage sites for I-Ltr,I-Gpi, I-Gze, I-MpeMI, I-PanMI, I-Cre, I-OnuI, I-HjeMI, and I-AniI (SeeTable 1) were generated by placing the endonuclease target site ofinterest placed in front of the mCherry fluorescent protein ORF in the+3 reading frame. HEK293T Reporter cell lines containinggenomically-integrated I-Ltr, I-Gpi, I-Gze, I-MpeMI, I-PanMI, I-Cre,I-OnuI, I-HjeMI, and I-AniI TLR reporter cassettes were then generated.Each cell line was transfected with an expression construct for itsrespective enzyme with or without co-transfection of an expressionconstruct encoding Trex2, and disruption rates were measured.

The effect of Trex2 co-expression with each of I-Ltr, I-Gpi, I-Gze,I-MpeMI, I-PanMI, I-Cre, I-OnuI, I-HjeMI, and I-AniI homingendonucleases was analyzed by flow cytometry. For each of the differentHoming Endonucleases tested, disruption rates increased when coupled toTrex2, demonstrating that the Trex2 exonuclease can facilitate genedisruption from breaks generated by a variety of different homingendonucleases, which leave different 3′ 4 bp overhangs and possessvarying enzyme kinetics. See FIG. 10. This data demonstrates that Trex2expression increases the mutagenesis rates associated with targeted DNAcleavage by a variety of homing endonucleases. Further, co-expression ofTrex2 with I-Gze increased mCherry+ expression significantly over thebackground levels observed with I-Gze expression alone. See FIG. 10.

Homing Endonucleases in the panel having very low activity were rescuedby coupling to Trex2. See FIG. 10. This suggests that HomingEndonucleases that appear inactive may be generating breaks at anundetectable rate, and that addition of Trex2 reveals these breaks bycatalyzing end processing prior to break ligation. This is consistentwith the observation that Trex2 can increase disruption rates of ahigher activity enzyme, such as I-SceI, even at very low expressionlevels.

To test the ability of Trex2 to reveal breaks caused by HomingEndonucleases having very low activity, the effect of coupling Trex2 onthe gene disruption rate of the I-AniI Homing Endonucleases was analyzedby flow cytometry. WT I-AniI exhibits very little activity in cells andexpression of WT I-AniI alone does not exhibit targeted disruptionactivity. See FIGS. 12A and 12B. Coupling of Trex2 to WT I-AniIincreases its gene disruption capacity to that of the highly activeI-AniI variant, I-AniI Y2. See FIGS. 12A and 12B. I-AniI Y2 wassubjected to several rounds of directed evolution to improve itsactivity. Coupling of Trex2 to an inactive form of I-AniI, I-AniI E148D,shows no increase in reporter expression. This data demonstrates thatTrex2 expression increases the mutagenesis rates associated withtargeted DNA cleavage by sub-active homing endonucleases.

Together, these results show that Trex2 can increase disruption ratesfor a variety of homing endonucleases and rescue low-activityendonucleases, effectively lowering the engineering bar for enzymesdesigned to produce gene disruption at novel target sites.

Example 3 Co-Expression of Trex2 Exonuclease Affects the Mutation RateAssociated with FokI Zinc Finger Nuclease Mediated Breaks

A reporter cell line was generated that harbors a 5′ ACC ATC TTC ttcaagGAC GAC GGC 3′ (SEQ ID NO. 147) target site for a corresponding zincfinger nuclease containing a FokI nuclease domain. Expression vectorsencoding the zinc finger nuclease were transduced into reporter celllines harboring the TLR-FokI reporter cassette with and without Trex2.Co-expression of Trex2 with the zinc finger nuclease results in anincreased mutation rate. See FIG. 11B.

Example 4 The Chimeric I-SceI-G4s-Trex2 Endo/Exo-Nuclease Fusion ProteinImproves the Rate of Targeted Disruption

Expression vectors comprising HA-I-SceI-BFP, (HA-I-SceI)-T2A-Trex2-BFPor (HA-I-SceI)-G4S-Trex2-BFP were constructed as described in Example 1.The I-SceI gene used to construct the expression vectors further encodedan N-terminal HA epitope tag. The (HA-I-SceI)-T2A-(HA-Trex2-BFP)expression vector expresses HA-I-SceI and Trex2 in a 1 to 1 ratio from asingle promoter, but the T2A linker sequence allows for two separateproteins to be produced from a single translation. The(HA-I-SceI)-G4S-(HA-Trex2)-BFP expression vector produces anendo/exo-nuclease fusion protein where HA-I-SceI and Trex2 proteins arecoupled together by a G4S linker peptide. The HA-I-SceI-BFP,(HA-I-SceI)-T2A-Trex2-BFP and (HA-I-SceI)-G4S-Trex2-BFP expressionvectors were transduced into HEK293 cells containing agenomically-integrated cassette corresponding to the targeted disruptionreporter illustrated in FIG. 1A.

Following transduction of the cell line with the expression vectors, thecells were analyzed for mCherry+ expression by flow cytometry. The plotshown in FIG. 3A demonstrated that I-SceI-G4S-Trex2 endo/exo fusionproteins are active and increase targeted disruption rates overprovision of I-SceI alone. See FIG. 3A-C.

However, Sce-G4S-Trex2, despite stable fusion protein expression, wasinferior at inducing gene disruption compared to Sce-T2A-Trex2, possiblydue to steric hindrance. See FIG. 3A.

An anti-HA western blot was performed to assess the stability of theHA-I-SceI, HA-I-SceI-T2A and (HA-I-SceI)-G4S-Trex2 proteins in theexpressing cells. As shown in FIGS. 3B and 3C, the chimeric(HA-I-SceI)-G4S-Trex2 endo-exo fusion protein was expressed at the samelevels as I-SceI alone, or I-SceI containing a residual T2A tag peptide.

Example 5 Co-Expression of I-SceI and Trex2 Exonuclease Increases theRate of I-SceI-Induced Mutations in Primary Cells

To determine if Trex2 would increase gene disruption rates in primarycells, primary murine embryonic fibroblasts (MEFs) were isolated from amouse with an I-SceI site “knocked into” the Interleukin-2 receptorsubunit gamma (IL2RG) locus (“Sce-SCID” mouse, unpublished data, G.C.,D.J.R., A.M.S). MEFs were isolated from Sce-SCID embryos at 12-14 daysgestation. Briefly, individual embryos were removed from the uterus andwashed with PBS. The head and red tissue were removed from the embryo,and the remaining tissue was minced. The tissue was incubated withtrypsin-EDTA for 10 minutes at 37° C., followed by centrifugation at10,000×G for 5 minutes. The pellet was re-suspended in MEF media andplated at 37° C. MEF cells were cultured in glutamine-free Dulbecco'smodified Eagle's medium supplemented with 2 mM L-glutamine, 10% FetalBovine Serum (FBS) and 1% penicillin/streptomycin.

1.0×10⁵ Sce-SCID MEF cells were seeded in a 24-well plate 24 hours priorto transduction with I-SceI or I-SceI.T2A.Trex2 expressing recombinantlentiviral vectors (LV). 0.5 μg DNA was used for each expression vector,and transfected using Fugene6 or XtremeGene9 (Roche) according to themanufacture's protocol. Cells were passaged 24 hours later and analyzed72 hours post transduction. Total gene disruption at the I-SceI targetsite was assayed using the digestion assay described in Example 1. A6-fold increase in disruption at the common gamma chain locus wasobserved with I-SceI coupled to Trex2 (I-SceI=15.8,I-SceI.T2A.Trex2=88.7). See FIGS. 9A and 9B. Additionally, since IL2RGis only expressed in a subset of differentiated hematopoietic cells,these experiments demonstrate Trex2 can facilitate high frequencydisruption at unexpressed loci.

Example 6 Effect of Exonuclease Over-Expression on Repair of EndogenousDNA Damage

To determine if exonuclease over-expression alters the cells ability torepair other types of endogenous DNA damage, Trex2 expressing cells aretreated with model DNA damage inducing agents. 1.0×106 Sce-SCID MEFswere seeded in a 10 cm dish 24 hours before transduction. 5000 μL of10×LV (pCVL.SFFV.sceD44A.IRES.BFP or T2A.TREX2.IRES.BFP) was added tothe culture with 4 μg/mL polybrene. 24 hours post-transduction, cellswere passaged to 15 cm plates. 72 hours post-transduction, 1.0×105Sce-SCID MEFs were seeded in a 12-well plate with 1 mL media and treatedas indicated with DNA damage inducing agents: Mitomycin C (SigmaAldrich, St. Louis), Camptothecin (Sigma Aldrich, St. Louis), orionizing radiation. 48 hours after exposure, cells were incubated in 0.5μg/mL PI as above and analyzed by flow cytometry. For CD34+ cells, 72hours post-transduction with Trex2 expressing LV, 2.0×10⁵ CD34+ HSCswere seeded in a 96-well plate in 200 μL of media, DNA damaging agentswere added to the media, and plates analyzed as above. Over-expressionof Trex2 had no adverse effect on cell cycle or sensitivity to model DNAdamaging agents, suggesting cells maintain high fidelity DNA repair atlesions occurring independently of those created by the endonuclease.See FIGS. 13, 14, 15A, 15B, 15C, 16A and 16B.

Example 7 Co-Expression of I-SceI and End-Processing Enzymes Increasesthe Rate of I-SceI-Induced Mutations

To determine if the results of coupling homing endonucleases with Trex2could be extended to other DNA modifying enzymes, a library of 13candidate enzymes possessing an array of biochemical end-processingactivities derived from mammalian, bacterial or viral species wasgenerated. See Table 2. The library of DNA end-processing enzymes wascloned into the pExodus vector with genes synthesized by Genscript(Piscataway, N.J.) as cDNA codon-optimized for human expression. See SEQID NOs. 110-145.

The library of DNA end-processing enzymes was screened by co-expressingeach enzyme with either the homing endonuclease, I-SceI, or the ZincFinger Nuclease, VF2468, in the respective HEK293T TLR cells. See FIGS.17A ₁, 17A₂, 17B, 18A, 19A and -19B. Five of DNA end-processing enzymes(Artemis, Tdt, Apollo, Rad2, and Exo1) robustly increased the genedisruption efficiency of I-SceI. See FIGS. 17A ₁, 17A₂ and 17B.Additionally, the gene disruption activity of these five enzymes wasanalyzed at three levels of I-SceI expression (quantified by the meanfluorescence intensity, MFI, of the BFP fluorophore). Coexpression ofthese enzymes with I-SceI increased I-SceI's mutagenic efficiency, evenat low levels of endonuclease expression. See FIGS. 18A and 18B. Incontrast, although several of the DNA end-processing enzymes possess 5′exonuclease activity, a significant effect of any enzyme on increasingthe gene disruption efficiency of the VF2468 ZFN was not observed. SeeFIG. 19A.

In addition, the library of DNA end-processing enzymes was screened byco-expressing each enzyme with TALEN. See FIG. 19B.

TABLE 2 Library of DNA End-Processing Enzymes. Species of NLS EnzymeGene name Activity origin added Reference Apollo SNM1B 5-3′ Human NoLenain, C. et al., The exonuclease Apollo 5′ exonuclease functionstogether with TRF2 to protect telomeresfrom DNA repair. Curr. Biol. 16,1303-1310 (2006). Artemis Artemis 5-3′ Human No Kurosawa, A., andexonuclease Adachi, N. Functions and regulation of Artemis: a goddess inthe maintenance ofgenome integrity. J Radiat. Res. (Tokyo) 51, 503-509(2010). Dna2 DNA2 5-3′ Human No Nimonkar, A. V., exonuclease, et al.BLM-DNA2- helicase RPA-MRN and EXO1-BLM-RPA- MRN constitute two DNA endresection machineries for human DNA break repair. Genes Dev 25, 350-362(2011). Exo1 EXO1 5-3′ Human No Nimonkar, A. V. exonuclease et al.BLM-DNA2- RPA-MRN and EXO1-BLM-RPA- MRN constitute two DNA end resectionmachineries for human DNA break repair. Genes Dev 25, 350-362 (2011).Orans, J., et al. Structures of human exonuclease 1 DNA complexessuggest a unified mechanism for nuclease family. Cell 145, 212-223(2011). Fen1 FEN1 5′ flap Human No Jagannathan, I., endonucleasePepenella, S. Hayes, J. J. Activity of FEN1 endonuclease on nucleosomesubstrates is dependent upon DNA sequence but not flap orientation. J.Biol. Chem. 286, 17521-17529 (2011). Tsutakawa, S. E., et al., Humanflap endonuclease structures, DNA double-base flipping, and a unifiedunderstanding of the FEN1 superfamily. Cell 145, 198-211 (2011). Mre11MRE11 5-3′ and 3-5′ Human No Garcia, V., Phelps, exonuclease S. E.,Gray, S., and Neale, M. J. Bidirectional resection of DNA double-strandbreaks by Mre11 and Exo1. Nature 479, 241-244 (2011). Rad2 n/a 5-3′Human No Lee, B. I., and (catalytic exonuclease Wilson, D. M., 3rddomain of (Exo1 catalytic The RAD2 domain Exo1) domain) of humanexonuclease 1 exhibits 5′ to 3′ exonuclease and flap structure-specificendonuclease activities. J Bio. l Chem. 274, 37763- 37769 (1999). TdT(terminal TdT Single- Human No Mahajan, K. N., et deoxynucleotidylstranded al., Association of transferase) Template terminal independentdeoxynucleotidyl DNA transferase with Ku. polymerase Proc. Natl. Acad.Sci. USA 96, 13926- 13931 (1999). RecE RecE 5-3′ E. coli Yes Zhang, J.,Xing, X., exonuclease Herr, A. B., and Bell, C. E. Crystal structure ofE. coli RecE protein reveals a toroidal tetramer for processingdouble-stranded DNA breaks. Structure 17, 690- 702 (2009). Lambda λ 5-3′Bacterioph Yes Zhang, J., McCabe, exonuclease exonuclease exonucleaseage λ K. A., and Bell, C. E. Crystal structures of lambda exonuclease incomplex withDNA suggest an electrostatic ratchet mechanism forprocessivity. Proc. Natl. Acad. Sci. USA 108, 11872-11877 (2011). Sox(T24I SOX 5-3′ alkaline Kaposi's Yes Glaunsinger, B., mutation)exonuclease sarcoma Chavez, L., and associated Ganem, D., The herpesexonuclease and host virus shutoff functions of the SOX protein ofKaposi′s sarcoma- associated herpesvirus are genetically separable. JVirol. 79, 7396-7401 (2005). Dahlroth, S. L., et al., Crystal structureof the shutoff and exonuclease protein from the oncogenic Kaposi′ssarcoma- associated herpes virus. FEBS J 276, 6636-6645 (2009). VacciniaDNA E9L 3-5′ Vaccinia Yes Gammon, D. B., and polymerase exonucleasepoxvirus Evans, D. H., The 3′- to-5′ exonuclease activity of vacciniavirus DNA polymerase is essential and plays a role in promoting virusgenetic recombination. J. Virol. 83, 4236-4250 (2009). UL-12 UL12 5-3′alkaline Herpes Yes Reuven, N. B., et al. exonuclease simplex The herpessimplex virus virus type 1 alkaline (HSV)-1 nuclease and single-stranded DNA binding protein mediate strand exchange in vitro. J. Virol.77, 7425-7433 (2003). Balasubramanian, N., et al. Physical interactionbetween the herpes simplex virus type 1 exonuclease, UL12, and the DNAdouble-strand break- sensing MRN complex. J. Virol. 84, 12504-12514(2010).

Example 8 Exonuclease Screen

An expression library containing both 3′ and 5′ specific exonucleases isscreened by expressing the exonucleases in cells containing a targeteddisruption reporter harboring a homing endonuclease target site, forexample an I-SceI target site. The exonucleases are co-expressed in thereporter cells with a homing endonuclease, for example I-Sce-I, whichgenerates 3′ overhangs upon cleaving its target site. Exonucleases whichincrease the rate of disruption, as visualized by mCherry+ expression,of the homing endonuclease target site over expression of the homingendonuclease alone are then identified.

An expression library containing both 3′ and 5′ specific exonucleases isadditionally screened by expressing the exonucleases in cells containinga targeted disruption reporter harboring a zinc finger endonucleasetarget site. The exonucleases are co-expressed in the reporter cellswith a zinc finger endonuclease, which generates 5′ overhangs uponcleaving its target site with Fok1. Exonucleases which increase the rateof disruption, as visualized by mCherry+ expression, of the zinc fingerendonuclease target site over expression of the zinc finger endonucleasealone are identified.

Example 9 Trex-Multiplex

Increasing disruption rates for individual nucleases by couplingendonuclease activity with exonuclease activity, enables multiplesimultaneous changes to a genome (multiplexing).

Three homing endonuclease are designed to knock out three differentgenes (x, y, and z). In the absence of exonuclease co-expression, theefficiency of producing a disruptive mutation, knockout, for each geneindividually is 10%, which means that the chance of successfullyproducing all three disruptive mutations in a single cell with a singleround of endonuclease expression is 0.1%. An exonuclease, for exampleTrex2, is co-expressed with the three homing endonucleases to increasethe rate of mutagenesis induced by the homing endonucleases. A 5-foldincrease in the mutagenesis rate, to 50% for each individual gene,improves the chance of disrupting all three in a single cell, in asingle round to 12.5%, a 125-fold difference.

Example 10 Reduction of Chromosomal Abnormalities During EndonucleaseMediated Targeted Disruption

Endonucleases, such as homing endonucleases, zinc finger nucleases, andTAL effector nucleases, induce indiscriminate chromosomal abnormalities,such as translocations. To test the ability of co-expression of anexonuclease that facilitates disruption of an endonuclease target siteto decrease the incidence of indiscriminate chromosomal abnormalities,an endonuclease, or a series of endonucleases are expressed in thepresence and absence of Trex2. Karyotyping analysis or GCH arrayanalysis is performed to determine if the incidence of genomicabnormalities induced by the endonucleases is reduced.

Example 11 Imparting Site-Specificity to Exonucleases

An exonuclease of interest, for example Trex2, is directly fused orcoupled through a linker peptide to an endonuclease or to a DNA bindingdomain which specifically binds to a target site adjacent to the sitewhere exonuclease activity is desired.

Example 12 Method of Treating Preventing, or Inhibiting HIV Infection ina Human Patient

Hematopoetic stem cells are isolated from bone marrow obtained from ahuman subject. The isolated stem cells are contacted with an effectiveamount of a zinc finger nuclease (ZFN) having target sites in the humanCCR-5 gene and contemporaneously contacted with a 5′ exonuclease. Thecontacted cells are allowed to recover in media for 72 hrs and thenscreened for targeted disruption of the CCR-5 gene. Cells containing atargeted disruption in CCR-5 are then propagated under appropriateconditions. The subject is given a daily intervenous (i.v.) injection ofabout 20 million cells containing the targeted disruption in the CCR-5gene. This dosage can be adjusted based on the results received and thejudgment of the attending physician. The protocol is preferablycontinued for at least about 1 or 2 weeks, preferably at least about 1or 2 months, and may be continued on a chronic basis.

Example 13 Method of Treating, Preventing, or Inhibiting HIV Infectionin a Human Patient

Hematopoetic stem cells are isolated from bone marrow obtained from ahuman subject. The isolated stem cells are contacted with an effectiveamount of a homing endonuclease engineered to cleave a target site inthe human CCR-5 gene and contemporaneously contacted with Trex2exonuclease. The contacted cells are allowed to recover in media for 72hrs and then screened for targeted disruption of the CCR-5 gene. Cellscontaining a targeted disruption in CCR-5 are then propagated underappropriate conditions. The subject is given a daily intervenous (i.v.)injection of about 20 million cells containing the targeted disruptionin the CCR-5 gene. This dosage can be adjusted based on the results ofthe treatment and the judgment of the attending physician. The protocolis preferably continued for at least about 1 or 2 weeks, preferably atleast about 1 or 2 months, and may be continued on a chronic basis.

Example 14 Method of Treating, Preventing, or Inhibiting HIV Infectionin a Human Patient

Hematopoetic stem cells are isolated from bone marrow obtained from ahuman subject. The isolated stem cells are contacted with an effectiveamount of a fusion protein comprising an endonuclease domain linked toan exonuclease domain wherein the endonuclease domain comprises a homingendonuclease engineered to cleave a target site in the human CCR-5 geneor fragment thereof and wherein the exonuclease domain comprises Trex2exonuclease or a fragment thereof. The contacted cells are allowed torecover in media for 72 hrs and then screened for targeted disruption ofthe CCR-5 gene. Cells containing a targeted disruption in CCR-5 are thenpropagated under appropriate conditions. The subject is given a dailyintervenous (i.v.) injection of about 20 million cells containing thetargeted disruption in the CCR-5 gene. This dosage can be adjusted basedon the results of the treatment and the judgment of the attendingphysician. The protocol is preferably continued for at least about 1 or2 weeks, preferably at least about 1 or 2 months, and may be continuedon a chronic basis.

Example 15 End-Modifying Enzyme Screen

An expression library containing end-modifying enzymes is screened byexpressing the end-modifying enzymes in cells containing a targeteddisruption reporter harboring a homing endonuclease target site, forexample an I-SceI target site. The end-modifying enzymes areco-expressed in the reporter cells with a homing endonuclease, forexample I-Sce-I, which generates 3′ overhangs upon cleaving its targetsite. End-modifying enzymes which increase the rate of disruption, asvisualized by mCherry+ expression, of the homing endonuclease targetsite over expression of the homing endonuclease alone are thenidentified.

An expression library containing end-modifying enzymes is additionallyscreened by expressing the exonucleases in cells containing a targeteddisruption reporter harboring a zinc finger endonuclease target site.The end-modifying enzymes are co-expressed in the reporter cells with azinc finger endonuclease, which generates 5′ overhangs upon cleaving itstarget site with Fok1. End-modifying enzymes which increase the rate ofdisruption, as visualized by mCherry+ expression, of the zinc fingerendonuclease target site over expression of the zinc finger endonucleasealone are identified.

Example 16 Method of Treating, Preventing, or Inhibiting Cancer in aHuman Patient

A patient having cancer is identified. The isolated an effective amountof an endonuclease targeting a site within the regulatory or codingsequence of an anti-apoptotic gene is administered in combination withan end processing enzyme. The patient is monitored for increasedapoptosis and or decreased malignant cell proliferation. In someembodiments, tumor growth is monitored. The protocol may be administeredon a periodic or chronic basis.

1.-30. (canceled)
 31. A method of increasing mutagenesis at adouble-strand DNA (dsDNA) break at a selected dsDNA target site in aeukaryotic cell comprising: selecting a dsDNA target site formutagenesis; selecting a homing endonuclease engineered to bind andcleave the selected dsDNA target site; and coupling the activity of thehoming endonuclease and the activity of a 3′ to 5′ exonuclease withinthe eukaryotic cell so as to increase mutagenesis at the selected dsDNAtarget site.
 32. The method of claim 31, wherein the dsDNA target siteis within a gene.
 33. The method of claim 31, wherein the dsDNA targetsite is within a non-coding sequence of a gene.
 34. The method of claim33, wherein the non-coding sequence is a regulatory sequence.
 35. Themethod of claim 34, wherein the regulatory sequence is a promoter,enhancer, or splice site.
 36. The method of claim 31, wherein the dsDNAtarget site is within a coding sequence of a gene.
 37. The method ofclaim 32, wherein the gene is CCR-5.
 38. The method of claim 32, whereinthe gene is Stat-3.
 39. The method of claim 31, wherein the eukaryoticcell is a yeast cell.
 40. The method of claim 31, wherein the eukaryoticcell is an algae cell.
 41. The method of claim 31, wherein theeukaryotic cell is a plant cell.
 42. The method of claim 31, wherein theeukaryotic cell is a mammalian cell.
 43. The method of claim 31, whereinthe mutagenesis is an insertion at the selected dsDNA target site. 44.The method of claim 31, wherein the mutagenesis is a deletion at theselected dsDNA target site.
 45. The method of claim 31, wherein thehoming endonuclease is engineered from a homing endonuclease selectedfrom the group consisting of: I-AniI, I-SceI, I-CeuI, PI-PspI, PI-Sce,I-SceIV, I-CsmI, I-Pant, I-PanII, I-PanMI, I-SceII, I-PpoI, I-SceIII,I-CreI, I-LtrI, I-GpiI, I-GzeI, I-OnuI, I-HjeMI, I-TevI, I-TevII, andI-TevIII.
 46. The method of claim 31, wherein the homing endonuclease isengineered from a homing endonuclease selected from the group consistingof: I-Ltr, I-Gpi, I-Gze, I-MpeMI, I-PanMI, I-CreI, I-OnuI, I-HjeMI, andI-AniI.
 47. The method of claim 31, wherein the homing endonuclease isengineered from I-SceI.
 48. The method of claim 31, wherein the homingendonuclease is engineered from I-CreI.
 49. The method of claim 31,wherein the homing endonuclease is engineered from I-OnuI.
 50. Themethod of claim 31, wherein the homing endonuclease is engineered fromI-Ltr.
 51. The method of claim 31, wherein the homing endonuclease isengineered from I-Gpi.
 52. The method of claim 31, wherein the homingendonuclease is engineered from I-Gze.
 53. The method of claim 31,wherein the homing endonuclease is engineered from I-MpeMI.
 54. Themethod of claim 31, wherein the homing endonuclease is engineered fromI-PanMI.
 55. The method of claim 31, wherein the homing endonuclease isengineered from I-HjeMI.
 56. The method of claim 31, wherein the homingendonuclease is engineered from I-AniI.
 57. The method of claim 31,wherein the 3′-5′ exonuclease is Trex2 or a biologically active fragmentthereof.
 58. The method of claim 31, wherein the 3-5′ exonuclease isTrex2.
 59. The method of claim 31, wherein the 3′-5′ exonuclease is abiologically active fragment of Trex2.
 60. The method of claim 31,wherein the homing endonuclease and the 3′-5′ exonuclease are encoded bya single polynucleotide.
 61. The method of claim 31, wherein the homingendonuclease is coupled to the 3′-5′ exonuclease by a linker domain. 62.The method of claim 61, wherein the linker domain is a chemical linker.63. The method of claim 61, wherein the linker domain is a peptidelinker comprising 4 to 30 amino acids.
 64. The method of claim 63,wherein the linker domain is a G4S linker.
 65. The method of claim 63,wherein the linker domain is a T2A linker.
 66. The method of claim 31,wherein the homing endonuclease is coupled to the 3′-5′ exonuclease byan IRES sequence.
 67. The method of claim 31, wherein the homingendonuclease is engineered from a homing endonuclease selected from thegroup consisting of: I-Ltr, I-Gpi, I-Gze, I-MpeMI, I-PanMI, I-CreI,I-OnuI, I-HjeMI, and I-AniI; the exonuclease is Trex2 or a biologicallyactive fragment thereof; and the homing endonuclease is coupled to the3′-5′ exonuclease by a peptide linker domain comprising 4 to 30 aminoacids
 68. A method of increasing mutagenesis at a double-strand DNA(dsDNA) break at a selected dsDNA target site in a eukaryotic cellcomprising: selecting a dsDNA target site for mutagenesis; selecting aI-CreI homing endonuclease engineered to bind and cleave the selecteddsDNA target site; and coupling the activity of the I-CreI homingendonuclease to the activity of a 3′ to 5′ exonuclease within theeukaryotic cell so as to increase mutagenesis at the selected dsDNAtarget site.
 69. A method of increasing mutagenesis at a double-strandDNA (dsDNA) break at a selected dsDNA target site in a eukaryotic cellcomprising: selecting a dsDNA target site for mutagenesis; selecting aI-SceI homing endonuclease engineered to bind and cleave the selecteddsDNA target site; and coupling the activity of the I-SceI homingendonuclease to the activity of a 3′ to 5′ exonuclease within theeukaryotic cell so as to increase mutagenesis at the selected dsDNAtarget site.
 70. A method of increasing mutagenesis at a double-strandDNA (dsDNA) break at a selected dsDNA target site in a eukaryotic cellcomprising: selecting a dsDNA target site for mutagenesis; selecting aI-OnuI homing endonuclease engineered to bind and cleave the selecteddsDNA target site; and coupling the activity of the I-OnuI homingendonuclease to the activity of a 3′ to 5′ exonuclease within theeukaryotic cell so as to increase mutagenesis at the selected dsDNAtarget site.
 71. The method of claim 68, wherein the 3′ to 5′exonuclease is Trex2 or a biologically active fragment thereof.
 72. Themethod of claim 71, wherein the 3′ to 5′ exonuclease is Trex2.
 73. Themethod of claim 71, wherein the 3′ to 5′ exonuclease is a biologicallyactive fragment of Trex2.
 74. The method of claim 68, wherein the I-CreIhoming endonuclease and the 3′ to 5′ exonuclease are encoded by a singlepolynucleotide.
 75. The method of claim 74, wherein the I-CreI homingendonuclease is coupled to the 3′ to 5′ exonuclease by a linker domain.76. The method of claim 75, wherein the linker domain is a chemicallinker.
 77. The method of claim 75, wherein the linker domain is apeptide linker comprising 4 to 30 amino acids.
 78. The method of claim77, wherein the linker domain is a G4S linker.
 79. The method of claim77, wherein the linker domain is a T2A linker.
 80. The method of claim74, wherein the I-CreI homing endonuclease is coupled to the exonucleaseby an IRES sequence.
 81. The method of claim 79, wherein the I-SceIhoming endonuclease and the 3′ to 5′ exonuclease are encoded by a singlepolynucleotide.
 82. The method of claim 81, wherein the I-SceI homingendonuclease is coupled to the 3′ to 5′ exonuclease by a linker domain.83. The method of claim 82, wherein the linker domain is a chemicallinker.
 84. The method of claim 82, wherein the linker domain is apeptide linker comprising 4 to 30 amino acids.
 85. The method of claim84, wherein the linker domain is a G4S linker.
 86. The method of claim84, wherein the linker domain is a T2A linker.
 87. The method of claim81, wherein the I-SceI homing endonuclease is coupled to the exonucleaseby an IRES sequence.
 88. The method of claim 70, wherein the I-OnuIhoming endonuclease and the 3′ to 5′ exonuclease are encoded by a singlepolynucleotide.
 89. The method of claim 88, wherein the I-OnuI homingendonuclease is coupled to the 3′ to 5′ exonuclease by a linker domain.90. The method of claim 89, wherein the linker domain is a chemicallinker.
 91. The method of claim 89, wherein the linker domain is apeptide linker comprising 4 to 30 amino acids.
 92. The method of claim91, wherein the linker domain is a G4S linker.
 93. The method of claim91, wherein the linker domain is a T2A linker.
 94. The method of claim88, wherein the I-OnuI homing endonuclease is coupled to the exonucleaseby an IRES sequence.